Biomolecule interaction using atomic force microscope

ABSTRACT

The present patent application describes a cantilever for atomic force microscopy (AFM), which includes a cantilever body having a fixed end and a free end, the free end having a surface region being chemically modified by a dendron in which a plurality of termini of the branched region of the dendron are bound to the surface, and a terminus of the linear region of the dendron is functionalized.

CONTINUING DATA

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 11/464,481, filed Aug. 14, 2006, which claimspriority to U.S. Provisional Patent Application No. 60/707,892, filedAug. 12, 2005, and U.S. Provisional Patent Application No. 60/817,608,filed Jun. 28, 2006, the contents of which are incorporated by referenceherein in their entirety. This application also claims priority toPCT/KR2005/002651, filed Aug. 12, 2005, which claims priority to U.S.Provisional Patent Application No. 60/601,237, filed Aug. 12, 2004, thecontents of which are incorporated by reference herein in theirentirety.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The present invention relates generally to atomic force microscopy(AFM), a cantilever for AFM, and an apparatus and a measuring method ofintermolecular interaction between the biomolecules using the same. Thepresent invention regards the usage of dendron coated Bio-AFM tips inmeasuring the interaction force between biomolecules. The presentinvention also provides details on Bio-AFM Force Mapping of cellreceptors by using surfaces with controlled meso spaces.

(b) Description of the Related Art

In the post-genomic era, quantitative and comprehensive studies ongenome for drug discovery, as well as disease diagnosis and prevention,are fast-growing research and development areas. Growth in these sectorshas already produced a strong demand for advanced biomolecularrecognition probes with high sensitivity and excellent specificity (K.Wang et al., Anal. Chem. 76, 5721 2004).

Out of many biomolecular recognition studies, understanding themechanical stability (or recognition property) of complementary DNAstrands is crucial for a profound understanding of numerous importantbiological processes, such as DNA transcription, gene expression andregulation, and DNA replication. In this respect, stretching andforce-induced melting of DNA have thus been investigated using severaltechniques, such as optical tweezers, micro-pipette suction, and AFM (H.Clausen-Schaumann, M. Seitz, R. Krautbauer, H. E. Gaub, Curr. Opin.Chem. Biol. 4, 524, 2000; R. Merkel, Physics Reports 346, 343, 2001; G.U. Lee, L. A. Chris, R. J. Colton, Science 266, 771, 1994).

As it is possible to measure specific interactions between individualmolecules at small length scales and high sensitivity down to forces ofa few piconewtons, AFM is becoming a rapidly developing technique forprobing affinity and recognition properties at the molecular level (R.Krautbauer, M. Rief, H. E. Gaub, Nano Lett. 3, 493, 2003). Compared withother sensitive methods for force measurements, AFM has the advantagesof high force resolution and high spatial resolution, and is operableunder physiological conditions for investigation of specificinteractions in biological processes, such as electrostatic interactions(J. Wang, A. J. Bard, Anal. Chem. 73, 2207, 2001), ligand-receptorbinding (S. M. Rigby-Singleton et al., J. Chem. Soc., Perkin Trans. 21722, 2002), antigen-antibody interactions (F. Schwesinger et al., Proc.Natl. Acad. Sci. U.S.A. 97, 9972, 2000), aptamer-protein interactions(C. Bai et al., Anal. Chem. 75, 2112, 2003), protein folding/unfolding(P. M. Williams et al., Nature 422, 446, 2003; M. S. Z. Kellermayer, S.B. Smith, H. L. Granzier, C. Bustamante, Science 276, 1112, 1997),cell-cell adhesion (M. Benoit, D. Gabriel, G. Gerisch, H. E. Gaub,Nature Cell Biol. 2, 313, 2000) and DNA-DNA hybridization (C. W. Frank,Biophys. J. 76, 2922, 1999).

Although many investigations have been performed on unbinding forcemeasurement between complementary DNA strands (H. Clausen-Schaumann, M.Seitz, R. Krautbauer, H. E. Gaub, Curr. Opin. Chem. Biol. 4, 524, 2000;R. Merkel, Physics Reports 346, 343, 2001; G. U. Lee, L. A. Chris, R. J.Colton, Science 266, 771. 1994; R. Krautbauer, M. Rief, H. E. Gaub, NanoLett. 3, 493, 2003), the recognition between the DNA strands during thestudies at the single molecular level is problematic. The typicalimmobilization approach suffered from multi-point interaction, andresolving out single molecular interactions has not been an easy task.In order to avoid the unwanted interaction, surface density was reducedby mixing with an inactive surfactant, but the approach resulted in lowrecognition efficiency leading to less reliable analysis. Therefore,commonly practiced surface chemistry for such immobilization such asoxide-silane and gold-thiol chemistry (T. Hugel, M. Seitz, Macromol.Rapid. Commun. 22, 989, 2001; W. K. Zhang, X. Zhang, Prog. Polym. Sci.28, 1271, 2003) has yet to be optimized to retrieve invaluablefundamental information on single DNA-DNA interaction during the forcemeasurement with AFM.

Atomic Force Microscopy has traditionally played an important role inunderstanding the various interaction mechanisms between biomoleculespresent inside organisms. Through its ability to analyze interactionforces, its importance within the fields of nano and biotechnology isexpected to increase into the future as more studies are conducted onthe molecular level.

Taking advantage of this technology, many efforts have been made toinvestigate interactive mechanisms between biomolecules on differentsurfaces. Unlike liquid, however, observation of biological material onsurfaces produce unique problems such as involuntary adsorption andsteric hindrance. Among the many measures taken to counter suchproblems, the most popular one involve measuring monomolecularinteraction by applying complex self-assembling film onto surfaces. Whenobserving the singular interaction force between two biomolecules withAFM, two issues come to the forefront. The first problem regards thedifference in biomolecular activity on a surface as opposed to withinthe body. The second issue hinges around the fact that monomolecularinteraction cannot be guaranteed in such a setting. Prior research hasshown that both problems can be addressed by using self-assembling filmtechnology and meso space manipulation technology (Langmuir 2005, 21,4257, WO 2006/016787) The technology involves observing intermolecularinteractions on the monomolecular level by controlling the spacing andnumber of biomolecules through limitation of the number of functionalgroups on which the molecules can be introduced. The most common problemof this method involves the unintended attraction between molecules ofthe same functional group, resulting in phase separation. In addition,there is no concrete evidence that the biomolecules are evenly spacedbetween each other.

The importance of biomolecular research using Bio-AFM technology isgrowing rapidly. Its ability to observe nonconductive material such asbiomolecules on the nanometer level in a liquid environment thatsupports biological activity has made Bio-AFM an important tool instudying the structure and substructures of biological molecules.Additionally, Bio-AFM enables close observation of molecular interactionby its Bio-AFM tip, onto which a biomolecule can be loaded. Importantapplications include observation of interactions between complementaryDNA molecules, mutual interactions between proteins, ligand-receptorinteractions, the latter of which holds significance in studyingimmunological responses to drugs. In researching interaction forcesbetween biomolecules on the monomolecular level, high sensitivity is ofprimary importance. Monomolecule observation can be accomplished throughmethods such as Bio-AFM, optical tweezing and magnetic tweezing. Eachmethod comes with its own shortcomings, such as loss of accuracy underthe magnetic tweezing method and potential damage that can be incurredupon molecules under the optical tweezing method.

In order to research ligand-receptor mechanisms using Bio-AFM, a ligandneeds to be loaded on top of the tip. Generally this is accomplished byutilizing biotin-streptavidin interactions or by use of compoundself-assembly films. However, such methods cannot directly controlmolecular distancing, and can cause ligands to concentrate in certainareas, posing difficulties in observing ligand-receptor interactionswith accuracy.

SUMMARY OF THE INVENTION

An object of the invention is to provide a cantilever for atomic forcemicroscopy (AFM) comprising a cantilever body having a fixed end and afree end, the free end having a surface region being chemically modifiedby a dendron in which a plurality of termini of the branched region ofthe dendron are bound to the surface, and a terminus of the linearregion of the dendron is functionalized.

Another object of the present invention is to provide the cantilever forAFM where the dendrons are spaced at regular intervals between about 0.1nm and about 100 nm between the linear functionalized groups. Inparticular, the dendrons may be spaced at regular intervals of about 10nm.

A further object of the present invention is to provide a method formanufacturing the cantilever, comprising (i) functionalizing the surfaceregion of the cantilever so that it will react with the termini of thedendrons; and (ii) contacting the dendrons to the surface region so thatthe termini and the surface form a bond.

An object of the present invention is to provide a method formanufacturing the cantilever, wherein a probe nucleotide, ligand for areceptor or a linker molecule linked to the probe nucleotide or ligandis fixed to the terminus of the linear region of dendrons, comprisingthe steps of i) removing protecting group from the terminus of thelinear region of the dendrons on the surface region; and ii) contactinga probe nucleotide, ligand for a receptor or a linker molecule linked tothe probe nucleotide or ligand to the terminus of the linear region ofthe dendrons on the substrate so that the probe nucleotide, ligand orthe linker molecule and the terminus form a bond, wherein the linkermolecule is a homo-bifunctional or hetero-bifunctional linker.

The present invention also provides an apparatus for measuring aninteraction between one probe nucleotide or ligand and one targetnucleotide or ligand binding partner such as its receptor by atomicforce microscopy, the apparatus comprising:

a cantilever having a fixed end and a free end, the free end having asurface region being chemically modified by a dendron in which aplurality of termini of the branched region of the dendron is bound tothe surface, and a terminus of the linear region of the dendron isattached to the probe nucleotide or ligand;

a substrate on which is immobilized a target nucleotide or ligandbinding partner;

a controller for adjusting the relative position and orientation of thecantilever and target nucleotide or ligand binding partner on asubstrate to cause an interaction between the probe nucleotide or ligandimmobilized on the dendron-modified surface region of the cantilever andthe target nucleotide or ligand binding partner immobilized on asubstrate; and

a detector for measuring a physical parameter associated with theinteraction between the probe nucleotide or ligand and the samplenucleic acid or ligand binding partner.

In an embodiment, the substrate to be immobilized by the targetnucleotide or ligand binding partner can be adopted by any kind of thesurface modification method in the art. Preferably, the substrate has adendron-modified surface.

A further object of the present invention is to provide a method ofassaying a target nucleotide or ligand binding partner for interactionwith a probe nucleotide or ligand, the method comprising the steps of:

(a) providing a cantilever having a fixed end and a free end, the freeend having a surface region being chemically modified by dendrons inwhich a plurality of termini of the branched region of the dendrons arebound to the surface, and a substrate;

(b) chemically modifying the substrate to immobilize a target nucleotideor ligand binding partner thereon;

(c) chemically modifying the dendron-modified surface region of thecantilever to immobilize a probe nucleotide or ligand;

(d) coupling the substrate and the cantilever to an apparatus thatincludes a controller for adjusting the relative position andorientation of the substrate and the cantilever to cause an interactionbetween the probe nucleotide or ligand immobilized on thedendron-modified surface region of the cantilever and the targetnucleotide or ligand binding partner immobilized on the substrate of thesample support member,

(e) controlling the relative position and orientation of the cantileverand the substrate to cause an interaction between the probe nucleotideor ligand and the target nucleotide or ligand binding partner; and

(f) measuring a physical parameter associated with the interactionbetween the probe nucleotide or ligand and the target nucleotide orligand binding partner.

In the above, the terminus of the branched region may be functionalizedwith —COZ, —NHR, —OR′, or —PR″3, wherein Z may be a leaving group,wherein R may be an alkyl, wherein R′ may be alkyl, aryl, or ether, andR″ may be H, alkyl, alkoxy, or O. In particular, COZ may be ester,activated ester, acid halide, activated amide, or CO-imidazoyl; R may beC1-C4 alkyl, and R′ may be C1-C4 alkyl. Further, in the substratedescribed above, the polymer may be a dendron. Still further, the linearregion of the polymer may include a spacer region. Also the spacerregion may be connected to the branched region via a first functionalgroup. Such first functional group may be without limitation —NH2, —OH,—PH3, —COOH, —CHO, or —SH. Still further, the spacer region may comprisea linker region covalently bound to the first functional group.

In the substrate and AFM cantilever, preferably AFM tip, the linkerregion may comprise a substituted or unsubstituted alkyl, alkenyl,alkynyl, cycloalkyl, aryl, ether, polyether, ester, or aminoalkyl group.Still further, the spacer region may comprise a second functional group.The second functional group may include without limitation, —NH2, —OH,—PH3, —COOH, —CHO, or —SH. The second functional group may be located atthe terminus of the linear region. Also, a protecting group may be boundto the terminus of the linear region. Such protecting group may be acidlabile or base labile.

In another embodiment of the invention, in the AFM cantilever asdescribed above, a probe ligand or nucleotide and/or a target ligandbinding partner or nucleotide may be bound to the terminus of the linearregion of the dendron. In particular, the target nucleotide and theprobe nucleotide may be DNA, RNA, PNA, aptamer, nucleotide analog, or acombination thereof. The target-specific ligand may include nucleotidesbut may be more generally thought of as a chemical compound,polypeptide, carbohydrate, antibody, antigen, biomimetics, nucleotideanalog, or a combination thereof.

Further, the distance between the ligands or nucleotides bound to thelinear region of the dendron may be from about 0.1 to about 100 nm.

In yet another embodiment of the invention, the substrate describedabove may be made of semiconductor, synthetic organic metal, syntheticsemiconductor, metal, alloy, plastic, silicon, silicate, glass, orceramic. In particular, the substrate may be without limitation a slide,particle, bead, micro-well, or porous material.

These and other objects of the invention will be more fully understoodfrom the following description of the invention, the referenced drawingsattached hereto and the claims appended hereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A a schematic view of a bio-AFM, and FIGS. 1B and 1C arephotographs of the bio-AFM.

FIG. 2A is a schematic drawing of a cantilever for AFM, and FIG. 2Bshows an enlarged view of the tip of AFM cantilever in accordance withthe exemplary embodiment of the present invention, FIG. 2C shows avariety of commercially available AFM tip.

FIG. 3 is a schematic drawing of the interface between the probe tip ofAFM and substrate target for measuring binding and unbinding forces withthe AFM methodology.

FIG. 4A is a histogram showing the force distribution for acomplementary 30-base pair with relatively narrow spacing at aretraction velocity of 110 nm/s, and FIG. 4B to FIG. 4C are directmeasurements of single unbinding force of complementary 30 base pairswith a retraction velocity of 540 nm/s.

FIG. 5A is a histogram showing for a complementary 30-base pair withrelatively broad spacing at a retraction velocity of 110 nm/s, and FIG.5B to FIG. 5C are measurements of binding force of a complementary 30base pair at a retract velocity of 110 nm/s.

FIG. 6A and FIG. 6B are a histogram showing the binding forcedistributions on complementary DNA duplexes, and FIG. 6C is a histogramshowing the unbinding force distributions on complementary DNA duplexes.

FIG. 7 is a histogram showing the binding force distributions for singlebase mismatched DNA duplexes.

FIG. 8 is a histogram showing the binding force distributions on doublebase mismatched DNA duplexes.

FIG. 9 shows a schematic drawing of a cantilever for an AFM and anenlarged view of a tip of an AFM cantilever.

FIG. 10 a shows the schematic view of the dendron-modified AFM tiptethered with ligands having enough spacing.

FIG. 10 b shows the schematic view of the AFM tip tethered with theclosely packed ligands.

FIG. 11 a shows the schematic drawing of the method of immobilizing aprotein on the dendron-modified AFM tip.

FIG. 11 b shows the schematic drawing of the method of immobilizing aprotein on the dendron-modified substrate.

FIG. 12 shows the schematic view of the method of force measurementusing an AFM

FIG. 13 a shows a scheme for the force measurement with a blockingprotein using an AFM.

FIG. 13 b shows a scheme for the force measurement with a competitiveprotein using an AFM.

FIG. 14 a is the histogram showing the force distribution for theinteraction between Munc-18-1 and PLD1-PX.

FIG. 14 b is the histogram showing the force distribution for theinteraction between Munc-18-1 and PLD1-PX after adding excess amounts offree Munc-18-1 in solution.

FIG. 15 is a graph showing the forces depending on the concentration ofthe competitive protein, PLC-γ1.

FIG. 16 a shows a schematic view of the method of screening thereceptors on a cell with a ligand-coated AFM tip.

FIGS. 16 b-16 d show the force curves for the interaction between FPR1and its biding peptide.

FIGS. 17 a-17 b show the force map and histogram for the interactionbetween FPR1 and its biding peptide on each different area of a cell.

FIG. 18 shows the force map and histogram for the interaction betweenFPR1 and its biding peptide before and after blocking with a free WKYMVm(SEQ ID NO:17).

DETAILED DESCRIPTION

In the present application, “a” and “an” are used to refer to bothsingle and a plurality of objects.

As used herein, “aptamer” means a single-stranded, partiallysingle-stranded, partially double-stranded or double-stranded nucleotidesequence, advantageously replicatable nucleotide sequence, capable ofspecifically recognizing a selected nonoligonucleotide molecule or groupof molecules by a mechanism other than Watson-Crick base pairing ortriplex formation.

As used herein, “biomimetic” means a molecule, group, multimolecularstructure or method that mimics a biological molecule, group ofmolecules, structure.

The term “dendrimer” is characterized by a core, at least one interiorbranched layer, and a surface branched layer (see Petar et al, Pages641-645, In Chem. in Britain, August 1994). A “dendron” is a species ofdendrimer having branches emanating from a focal point, which is or canbe joined to a core, either directly or through a linking moiety to forma dendrimer. Many dendrimers include two or more dendrons joined to acommon core. However, the term “dendrimer” may be used broadly toencompass a single dendron.

As used herein, “hyperbranched” or “branched” as it is used to describea macromolecule or a dendron structure is meant to refer to a pluralityof polymers having a plurality of termini which are able to bindcovalently or ionically to a substrate. In one embodiment, themacromolecule containing the branched or hyperbranched structure is“pre-made” and is then attached to a substrate.

As used herein, “immobilized” means insolubilized or comprising,attached to or operatively associated with an insoluble, partiallyinsoluble, colloidal, particulate, dispersed, suspended and/ordehydrated substance or a molecule or solid phase comprising or attachedto a solid support.

As used herein, “library” refers to a random or nonrandom mixture,collection or assortment of molecules, materials, surfaces, structuralshapes, surface features or, optionally and without limitation, variouschemical entities, monomers, polymers, structures, precursors, products,modifications, derivatives, substances, conformations, shapes, orfeatures.

As used herein, “ligand” means a selected molecule capable ofspecifically binding to another molecule by affinity-based attraction,which includes complementary base pairing. Ligands include, but are notlimited to, nucleic acids, various synthetic chemicals, receptoragonists, partial agonists, mixed agonists, antagonists,response-inducing or stimulus molecules, drugs, hormones, pheromones,transmitters, autacoids, growth factors, cytokines, prosthetic groups,coenzymes, cofactors, substrates, precursors, vitamins, toxins,regulatory factors, antigens, haptens, carbohydrates, molecular mimics,structural molecules, effector molecules, selectable molecules, biotin,digoxigenin, crossreactants, analogs, competitors or derivatives ofthese molecules as well as library-selected nonoligonucleotide moleculescapable of specifically binding to selected targets and conjugatesformed by attaching any of these molecules to a second molecule.

As used herein, “ligand binding partner” refers to a moleculespecifically binds to the ligand.

As used herein, “linker molecule” and “linker” when used in reference toa molecule that joins the branched portion of a size-controlledmacromolecule such as a branched/linear polymer to a protecting group ora ligand. Linkers may include, for instance and without limitation,spacer molecules, for instance selected molecules capable of attaching aligand to a dendron.

As used herein, “low density” refers to about 0.01 to about 0.5probe/nm2, preferably about 0.05 to about 0.2, more preferably about0.075 to about 0.15, and most preferably about 0.1 probe/nm2.

As used herein, “molecular mimics” and “mimetics” are natural orsynthetic nucleotide or nonnucleotide molecules or groups of moleculesdesigned, selected, manufactured, modified or engineered to have astructure or function equivalent or similar to the structure or functionof another molecule or group of molecules, e.g., a naturally occurring,biological or selectable molecule. Molecular mimics include moleculesand multimolecular structures capable of functioning as replacements,alternatives, upgrades, improvements, structural analogs or functionalanalogs to natural, synthetic, selectable or biological molecules.

As used herein, “probe nucleotide” or “target nucleotide” includes asequence of nucleotides, such as an oligonucleotide, and is not limitedto one nucleotide.

As used herein, “nucleotide analog” refers to molecules that can be usedin place of naturally occurring bases in nucleic acid synthesis andprocessing, preferably enzymatic as well as chemical synthesis andprocessing, particularly modified nucleotides capable of base pairingand optionally synthetic bases that do not comprise adenine, guanine,cytosine, thymidine, uracil or minor bases. This term includes, but isnot limited to, modified purines and pyrimidines, minor bases,convertible nucleosides, structural analogs of purines and pyrimidines,labeled, derivatized and modified nucleosides and nucleotides,conjugated nucleosides and nucleotides, sequence modifiers, terminusmodifiers, spacer modifiers, and nucleotides with backbonemodifications, including, but not limited to, ribose-modifiednucleotides, phosphoramidates, phosphorothioates, phosphonamidites,methyl phosphonates, methyl phosphoramidites, methyl phosphonamidites,5′-β-cyano ethyl phosphoramidites, methylenephosphonates,phosphorodithioates, peptide nucleic acids, achiral and neutralinternucleotidic linkages and nonnucleotide bridges such as polyethyleneglycol, aromatic polyamides and lipids.

As used herein, “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The term may also include variants on the traditional peptidelinkage joining the amino acids making up the polypeptide.

As used herein, “protecting group” refers to a group that is joined to areactive group (e.g., a hydroxyl or an amine) on a molecule. Theprotecting group is chosen to prevent reaction of the particular radicalduring one or more steps of a chemical reaction. Generally theparticular protecting group is chosen so as to permit removal at a latertime to restore the reactive group without altering other reactivegroups present in the molecule. The choice of a protecting group is afunction of the particular radical to be protected and the compounds towhich it will be exposed. The selection of protecting groups is wellknown to those of skill in the art. See, for example Greene et al.,Protective Groups in Organic Synthesis, 2nd ed., John Wiley & Sons, Inc.Somerset, N.J. (1991), which is incorporated by reference herein in itsentirety.

As used herein, “protected amine” refers to an amine that has beenreacted with an amino protecting group. An amino protecting groupprevents reaction of the amide function during attachment of thebranched termini to a solid support in the situation where the lineartip functional group is an amino group. The amino protecting group canbe removed at a later time to restore the amino group without alteringother reactive groups present in the molecule. For example, theexocyclic amine may be reacted with dimethylformamide diethylacetal toform the dimethylaminomethylenamino function. Amino protecting groupsgenerally include carbamates, benzyl radicals, imidates, and othersknown to those of skill in the art. Preferred amino protecting groupsinclude, but are not limited to, p-nitrophenylethoxycarbonyl ordimethyaminomethylenamino.

As used herein, “regular intervals” refers to the spacing between thetips of the size-controlled macromolecules, which is a distance fromabout 1 nm to about 100 nm so as to allow room for interaction betweenthe target-specific ligand and the target substantially///without sterichindrance. Thus, the layer of macromolecules on a substrate is not toodense for specific molecular interactions to occur.

As used herein, “solid support” refers to a composition comprising animmobilization matrix such as, but not limited to, insolubilizedsubstance, solid phase, surface, substrate, layer, coating, woven ornonwoven fiber, matrix, crystal, membrane, insoluble polymer, plastic,glass, biological or biocompatible or bioerodible or biodegradablepolymer or matrix, microparticle or nanoparticle. Solid supportsinclude, for example and without limitation, monolayers, layers,commercial membranes, resins, matrices, fibers, separation media,chromatography supports, polymers, plastics, glass, mica, gold, beads,microspheres, nanospheres, silicon, gallium arsenide, organic andinorganic metals, semiconductors, insulators, microstructures andnanostructures. Microstructures and nanostructures may include, withoutlimitation, microminiaturized, nanometer-scale and supramolecularprobes, tips, bars, pegs, plugs, rods, sleeves, wires, filaments, andtubes.

As used herein, “spacer molecule” refers to one or more nucleotideand/or nonnucleotide molecules, groups or spacer arms selected ordesigned to join two nucleotide or non-nucleotide molecules andpreferably to alter or adjust the distance between the two nucleotide ornon-nucleotide molecules.

As used herein, “specific binding” refers to a measurable andreproducible degree of attraction between a ligand and its specificbinding partner or between a defined sequence segment and a selectedmolecule or selected nucleic acid sequence. The degree of attractionneed not be maximized to be optimal. Weak, moderate or strongattractions may be appropriate for different applications. The specificbinding which occurs in these interactions is well known to thoseskilled in the art. When used in reference to synthetic defined sequencesegments, synthetic aptamers, synthetic heteropolymers, nucleotideligands, nucleotide receptors, shape recognition elements, andspecifically attractive surfaces. The term “specific binding” mayinclude specific recognition of structural shapes and surface features.Otherwise, specific binding refers explicitly to the specific,saturable, noncovalent interaction between two molecules (i.e., specificbinding partners) that can be competitively inhibited by a thirdmolecule (i.e., competitor) sharing a chemical identity (i.e., one ormore identical chemical groups) or molecular recognition property (i.e.,molecular binding specificity) with either specific binding partner. Thecompetitor may be, e.g., a crossreactant, or analog of an antibody orits antigen, a ligand or its receptor, or an aptamer or its target.Specific binding between an antibody and its antigen, for example, canbe competitively inhibited either by a crossreacting antibody or by acrossreacting antigen. The term “specific binding” may be used forconvenience to approximate or abbreviate a subset of specificrecognition that includes both specific binding and structural shaperecognition.

As used herein, “substrate,” when used in reference to a substance,structure, surface or material, means a composition comprising anonbiological, synthetic, nonliving, planar, spherical or flat surfacethat is not heretofore known to comprise a specific binding,hybridization or catalytic recognition site or a plurality of differentrecognition sites or a number of different recognition sites whichexceeds the number of different molecular species comprising thesurface, structure or material. The substrate may include, for exampleand without limitation, semiconductors, synthetic (organic) metals,synthetic semiconductors, insulators and dopants; metals, alloys,elements, compounds and minerals; synthetic, cleaved, etched,lithographed, printed, machined and microfabricated slides, devices,structures and surfaces; industrial polymers, plastics, membranes;silicon, silicates, glass, metals and ceramics; wood, paper, cardboard,cotton, wool, cloth, woven and nonwoven fibers, materials and fabrics;nanostructures and microstructures unmodified by immobilization probemolecules through a branched/linear polymer.

As used herein, “target-probe binding” means two or more molecules, atleast one being a selected molecule, attached to one another in aspecific manner. Typically, a first selected molecule may bind to asecond molecule that either indirectly, e.g., through an interveningspacer arm, group, molecule, bridge, carrier, or specific recognitionpartner, or directly, i.e., without an intervening spacer arm, group,molecule, bridge, carrier or specific recognition partner,advantageously by direct binding. A selected molecule may specificallybind to a nucleotide via hybridization. Other noncovalent means forconjugation of nucleotide and nonnucleotide molecules include, e.g.,ionic bonding, hydrophobic interactions, ligand-nucleotide binding,chelating agent/metal ion pairs or specific binding pairs such asavidin/biotin, streptavidin/biotin, anti-fluorescein/fluorescein,anti-2,4-dinitrophenol (DNP)/DNP, anti-peroxidase/peroxidase,anti-digoxigenin/digoxigenin or, more generally, receptor/ligand. Forexample, a reporter molecule such as alkaline phosphatase, horseradishperoxidase, β-galactosidase, urease, luciferase, rhodamine, fluorescein,phycoerythrin, luminol, isoluminol, an acridinium ester or a fluorescentmicrosphere which is attached, e.g., for labeling purposes, to aselected molecule or selected nucleic acid sequence using avidin/biotin,streptavidin/biotin, anti-fluorescein/fluorescein,anti-peroxidase/peroxidase, anti-DNP/DNP, anti-digoxigenin/digoxigeninor receptor/ligand (i.e., rather than being directly and covalentlyattached) may be conjugated to the selected molecule or selected nucleicacid sequence by means of a specific binding pair.

By controlling the spacing between immobilized DNA strands on thesurfaces of both an AFM tip and a substrate, unbinding and bindingforces of a single oligonucleotide were measured. It was observed thatthe recognition efficiency could be improved, and multiple and/orsecondary interaction was eliminated with appropriate choice of thespacing. In particular, histograms of unbinding force of DNA duplexeswith 20, 30, 40, and 50 base pairs became sharp and represented theforce of single duplex. Surprisingly, binding events were also observed,and the corresponding force coincided with the unbinding force. Also,linear increases of both forces were observed with the increase of DNAstrand length, and the force measurement was sensitive enough todiscriminate a single point mutation.

The present invention provides a cantilever for atomic force microscopy(AFM) comprising a cantilever body having a fixed end and a free end,the free end having a surface region being chemically modified by adendron in which a plurality of termini of the branched region of thedendron are bound to the surface, and a terminus of the linear region ofthe dendron is functionalized.

In an embodiment of the present invention, at least a tapered protrusionis provided in the vicinity of the free end of the cantilever, and theprotrusion is pyramidal or conical. Numerous analogous structures of theprobe tip are shown in FIG. 2C. Thus, the surface region of the free endof the cantilever is brought into contact with or into proximity with aparticular protrusion so that interactions between a molecule of thereference compound and a can be measured. All types of cantilevers forAFM can be used in the present invention, and they are not specificallylimited. The cantilever of the present invention can be used for alltype of AFM such as apparatus shown in FIGS. 1B and 1C. FIG. 1A shows anexample of a general atomic force microscope, and FIG. 2A is acantilever for AFM. The AFM of the present invention can be illustratedin reference to FIG. 1A. The AFM system 10 includes a base 15, frame 20having an opening on its central position fixed to the base 15, andtube-like piezoelectric actuator 55 fixed to the base 15. The tube-likepiezoelectric actuator 55 is deflectable in the vertical directionindicated by an arrow V2, i.e., in the direction of thickness of thecantilever by applying a voltage to the piezoelectric actuator from acontroller CO through wiring lines.

In reference to FIG. 2A, the cantilever 50 has a structure such that apiezoelectric actuator 25 is formed on one side of a substrate 95. Anexemplary embodiment of the cantilever, the cantilever 50 includes acantilever base 90 which has an electrode 10 formed on a insulatinglayer 110 laminated on rectangular substrate 95.

The cantilever may be constructed of any material known in the art foruse in AFM cantilevers, including Si, SiO₂, Si₃N₄, Si₃N₄Ox, Al, orpiezoelectric materials. The chemical composition of the cantilever isnot critical and is preferably a material that can be easilymicrofabricated and that has the requisite mechanical properties for usein AFM measurements. Likewise, the cantilever may be in any size andshape known in the art for AFM cantilevers. The size of the cantileverpreferably ranges from about 5 microns to about 1000 microns in length,from about 1 micron to about 100 microns in width, and from about 0.04microns to about 5 microns in thickness. Typical AFM cantilevers areabout 100 microns in length, about 20 microns in width and about 0.3microns in thickness. The fixed end of the cantilever may be adapted sothat the cantilever fits or interfaces with a cantilever-holding portionof a conventional AFM.

The surface region of the free end of the cantilever may be modified fortreatment with dendron for example, with siliane agents such as GPDES orTPU.

The apparatus and methods of the present invention are not limited touse with cantilever-based AFM instruments.

Polymers such as that in Chemical Formula 1 may be referred to indescribing the inventive polymer.

Various R, T, W, L, and X group variables are noted in chemicalformula 1. The polymer may comprise any branched or hyperbranched,symmetrical or asymmetrical polymer. The branched termini of the polymerbind to the substrate preferably by a plurality of termini. The linearend of the polymer may end with a functional group to which a protectinggroup or a target nucleotide may be attached. The distance between theprobes among the plurality of polymers on a substrate may be from about0.1 nm to about 100 nm, preferably about 1 nm to about 100 nm, morepreferably about 2 nm to about 70 nm, even more preferably about 2 nm toabout 60 nm, and most preferably about 2 nm to about 50 nm.

R-Groups

In Formula I, the polymer generally includes a branched section, whereinthe termini of the ends are functionalized to bind to a substrate.Within this branched section, the first generation group of branches Rx(R1, R2, R3) is connected to a second generation group of branchesR_(XX) (R11, R12, R13, R21, R22, R23, R31, R32, R33) by a functionalgroup, W. The second gene ration group of branches is connected to athird generation group of branches Rxxx (R111, R112, R113, R121, R122,R123, R131, R132, R133, R211, R212, R213, R221, R222, R223, R231, R232,R233, R311, R312, R313, R321, R322, R323, R331, R332, R333) by afunctional group W. And a further fourth generation may be connected tothe third generation branches in like fashion. The terminal R group isfunctionalized so that it is capable of binding to the substrate.

The R groups of all generations may be the same or different. Typically,the R group may be a repeating unit, a linear or branched organicmoiety, such as but not limited to alkyl, alkenyl, alkynyl, cycloalkyl,aryl, ether, polyether, ester, aminoalkyl, and so on. However, it isalso understood that not all of the R groups need to be the samerepeating unit. Nor do all valence positions for the R group need befilled with a repeating unit. For instance, in the first generationbranch, R_(x), R₁, R₂, R₃ all of the R groups at this branch level maybe the same repeating units. Or, R₁ may be a repeating unit, and R₂ andR₃ may be H or any other chemical entity. Or, R₂ may be a repeatingunit, and R₁ and R₃ may be H or any other chemical entity. Likewise, forthe second and third generation branches, any R group may be a repeatingunit, H or any other chemical entity.

Thus, a variety of shapes of polymers may be made in this way, forinstance, if R₁, R₁₁, R₁₁₁, R₁₁₂ and R₁₁₃ are the same repeating units,and all other R groups are H's or any number of small neutral moleculeor atom, then a fairly long and thin polymer having a branch with threefunctional group termini for R₁₁₁, R₁₁₂ and R₁₁₃ is made. A variety ofother optional chemical configurations are possible. Thus, it ispossible to obtain from about 3 to about 81 termini having a functionalgroup capable of binding to a substrate. A preferable number of terminimay be from about 3 to about 75, from about 3 to about 70, from about 3to about 65, from about 3 to about 60, from about 3 to about 55, fromabout 3 to about 50, from about 3 to about 45, from about 3 to about 40,from about 3 to about 35, from about 3 to about 30, from about 3 toabout 27, from about 3 to about 25, from about 3 to about 21, from about3 to about 18, from about 3 to about 15, from about 3 to about 12, fromabout 3 to about 9, or from about 3 to about 6.

T-Terminal Group

Terminal groups, T, are functional groups that are sufficiently reactiveto undergo addition or substitution reactions. Examples of suchfunctional groups include without limitation, amino, hydroxyl, mercapto,carboxyl, alkenyl, allyl, vinyl, amido, halo, urea, oxiranyl,aziridinyl, oxazolinyl, imidazolinyl, sulfonato, phosphonato,isocyanato, isothiocyanato, silanyl, and halogenyl.

W-Functional Group

In Formula I, W may be any functional group that may link a polymer toanother (or any other divalent organic) moiety, such as but not limitedto ether, ester, amide, ketone, urea, urethane, imide, carbonate,carboxylic acid anhydride, carbodiimide, imine, azo group, amidine,thiocarbonyl, organic sulphide, disulfide, polysulfide, organicsulphoxide, sulphite, organic sulphone, sulphonamide, sulphonate,organic sulphate, amine, organic phosphorous group, alkylen,alkyleneoxide, alkyleneamine and so on.

L-Spacer or Linker Group

In Chemical Formula 1, the linear portion of the polymer may include aspacer domain comprised of a linker region optionally interspersed withfunctional groups. The linker region may be comprised of a variety ofpolymers. The length of the linker may be determined by a variety offactors, including the number of branched functional groups binding tothe substrate, strength of the binding to the substrate, the type of Rgroup that is used, in particular, the type of repeating unit that isused, and the type of the protecting group or target nucleotide that isto be attached at the apex of the linear portion of the polymer.Therefore, it is understood that the linker is not to be limited to anyparticular type of polymer or to any particular length.

However, as a general guideline, the length of the linker may be fromabout 0.5 nm to about 20 nm, preferably, about 0.5 nm to about 10 nm,and most preferably about 0.5 nm to about 5 nm.

The chemical construct of the linker may include without limitation, alinear or branched organic moiety, such as but not limited tosubstituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, aryl, ether, polyether, ester, aminoalkyl, polyalkyleneglycol and so on. The linker may further include functional groups suchas those described above, and as such is not limited to any particularstructure. The linker group functionalized at the tip may comprise aprotective group.

X-Protecting Group

The choice of protecting group depends on numerous factors such as thedesirability of acid- or base-lability. Therefore, the invention is notlimited to any particular protecting group so long as it serves thefunction of preventing the reaction of the functional group with anotherchemical entity, and that it is capable of being stripped under desiredspecified conditions. A list of commercially available protecting groupsmay be found in the Sigma-Aldrich (2003) Catalog, the contents of whichas it relates to the disclosure of protective groups is incorporated byreference herein in its entirety.

The polymer may be deprotected, either in succession or in a singleoperation. Removal of the polypeptide and deprotection can beaccomplished in a single operation by treating the substrate-boundpolypeptide with a cleavage reagent, for example thianisole, water,ethanedithiol and trifluoroacetic acid.

In general, in one aspect of the invention, the protecting groups usedin the present invention may be those that are used in the sequentialaddition of one or more amino acids or suitably protected amino acids toa growing peptide chain.

Normally, either the amino or carboxyl group of the first amino acid isprotected by a suitable protecting group.

In a particularly preferred method, the amino function is protected byan acid or base sensitive group. Such protecting groups should have theproperties of being stable to the conditions of linkage formation, whilebeing readily removable without destruction of the growingbranched/linear polymer. Such suitable protecting groups may be withoutlimitation 9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl(Boc), benzyloxycarbonyl (Cbz), biphenylisopropyl-oxycarbonyl,t-amyloxycarbonyl, isobornyloxycarbonyl,(a,a)-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl,2-cyano-t-butyloxycarbonyl, and the like.

Particularly preferred protecting groups also include2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), p-toluenesulfonyl,4-methoxybenzenesulfonyl, adamantyloxycarbonyl, benzyl,o-bromobenzyloxycarbonyl, 2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu),cyclohexyl, cyclophenyl and acetyl (Ac), 1-butyl, benzyl andtetrahydropyranyl, benzyl, p-toluenesulfonyl and 2,4-dinitrophenyl.

In the addition method, the branched termini of the linear/branchedpolymer is attached to a suitable solid support. Suitable solid supportsuseful for the above synthesis are those materials which are inert tothe reagents and reaction conditions of the stepwisecondensation-deprotection reactions, as well as insoluble in the mediaused.

The removal of a protecting group such as Fmoc from the linear tip ofthe branched/linear polymer may be accomplished by treatment with asecondary amine, preferably piperidine. The protected portion may beintroduced in about 3-fold molar excess and the coupling may bepreferably carried out in DMF. The coupling agent may be withoutlimitationO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.).

The polymer may be deprotected, either in succession or in a singleoperation. Removal of the polypeptide and deprotection can beaccomplished in a single operation by treating the substrate-boundpolypeptide with a cleavage reagent, for example thianisole, water,ethanedithiol and trifluoroacetic acid.

The substrate may be any solid surface to which the branched/linearpolymer may bind through either covalent or ionic bond. The substratemay be functionalized so that binding may occur between the branchedtermini of the branched/linear polymer. The surface of the substrate maybe a variety of surfaces according to the needs of the practitioner inthe art. Preferably, the substrate may be a glass slide. Othersubstrates may include membrane filters such as but not limited tonitrocellulose or nylon. The substrate may be hydrophilic or polar, andmay possess negative or positive charge before or after coating.

The type of dendron and its preparation method is specifically disclosedin WO2005/026191, which is incorporated herein by reference.

Reaction scheme 1 is a scheme showing the synthesis of a dendron.Various starting materials, intermediate compounds, and dendroncompounds can be used, wherein “X” may be any protecting group,including anthracenemethyl (A), Boc, Fmoc, Ns and so forth.

A second generation branch dendron having surface reactive functionalgroups at the branch termini may be used, which self assembles andprovides appropriate spacing among themselves. Previous studies showedthat multiple ionic attractions between cations on a glass substrate andanionic carboxylates at the dendron's termini successfully generated awell-behaved monolayer, and guaranteed an inter-ligand space of over 24Å (Hong et al., Langmuir 19, 2357-2365 (2003)). To facilitatedeprotection and increase the deprotected apex amine's reactivity, thestructure was modified. Also, covalent bond formation between thedendron's carboxylic acid groups and the surface hydroxyl groups is aseffective as ionic attraction, while also providing enhanced thermalstability. Moreover, an oligoetheral interlayer was effective forsuppressing non-specific oligonucleotide binding.

The hydroxylated substrate was prepared by using a previously reportedmethod (Maskis et al., Nucleic Acids Res. 20, 1679-1684 (1992)).Substrates including oxidized silicon wafer, fused silica, and glassslide, were modified with (3-glycidoxypropyl)methyldiethoxysilane(GPDES) and ethylene glycol (EG). The dendron was introduced to theabove substrates through a coupling reaction between the dendron'scarboxylic acid group and the substrate's hydroxyl group using1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) or1-3-dicyclohexylcarbodiimide (DCC) in the presence of4-dimethylaminopyridine (DMAP) (Boden et al., J. Org. Chem. 50,2394-2395 (1985); Dhaon et al., J. Org. Chem. 47, 1962-1965 (1982)). Theincrease in thickness after dendron introduction was 11±2 Å, which wascomparable to the previous value observed for the ionic bonding (Hong etal., Langmuir 19, 2357-2365 (2003)).

After modification with di(N-succinimidyl) carbonate (DSC) according toa previously established method (Beier et al., Nucleic Acids Res. 27,1970-1977 (1999)), probe oligonucleotides were immobilized onto theactivated surface of glass slide by spotting 50 mM sodium bicarbonatebuffer (10% dimethylsulfoxide (DMSO), pH 8.5) solution of theappropriate amine-tethered oligonucleotide (20, uM) using a Microsys5100 Microarrayer (Cartesian Technologies, Inc.) in a class 10,000 cleanroom. Typically, for substrates with a reactive amine surface group, athiol-tethered oligonucleotide and a heterobifunctional linker such assuccinimidyl 4-maleimido butyrate (SMB) orsulphosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate(SSMCC) are employed (Oh et al., Langmuir 18, 1764-1769 (2002); Frutoset al., Langmuir 16, 2192-2197 (2000)). In contrast, because thedendron-modified surface guarantees a certain distance among the aminefunctional groups, use of homobifunctional linkers such as DSC is notproblematic. As a result, an amine-tethered oligonucleotide can beutilized for spotting. Unless cost effectiveness is important, use ofeasily oxidized thiol-tethered oligonucleotide should be avoided,although it is possible that such thiol-tethered oligonucleotides may beuseful under certain conditions.

To improve the recognition efficiency between complementary DNA strandsat the single molecular level, DNA oligomers was immobilized onto ananoscale-controlled dendron surface. The surface seemed to be ideal toincrease the efficiency since the mesospacing existing in the dendronrelieved the immobilized DNA from the steric hindrance (B. J. Hong, S.J. Oh, T. O. Youn, S. H. Kwon, J. W. Park, Langmuir 21, 4257, 2005).Either glycidylpropyldiethoxymethylsilane (or GPDES) orN-(3-(triethoxysilyl)propyl)-O-polyethyleneoxide urethane (or TPU) wasemployed to generate a sublayer, and the dendron (9-anthrylmethylN-({[tris({2-[({tris[(2-carboxyethoxy)methyl]methyl}amino)carbonyl]ethoxy}methyl)methyl]amino}carbonyl)propylcarbamate)(or 9-acid) was immobilized onto them. Previously, mesospacing betweenthe dendrons on the GPDES-modified surface was 32 Å on average (B. J.Hong, S. J. Oh, T. O. Youn, S. H. Kwon, J. W. Park, Langmuir 21, 4257,2005). In the case of TPU, an absorption peak observed at 257 nm arisingfrom the anthracene moiety of the pristine dendron was one half of thatin the GPDES case. Therefore it is suggested that the spacing of thedendron is larger than 32 Å in the TPU case. After deprotection of theanthracene protecting group, the amine group was activated withdi(N-succinimidyl)carbonate, and eventually an amine tetheringoligonucleotide was immobilized.

To understand the effect of the spacing, the two types of modificationwere employed for the substrate, while spacing on AFM tip was fixed withuse of 9-acid/TPU. Since estimation of the spring constant was believedto give a typical error of 10˜20%, the force was measured under variousconditions with an identical tip (FIG. 3). For example, force curveswere obtained for a complementary 30-base DNA (Table 1) immobilized on9-acid/GPDES substrate at various loading rates in the range between 110nm/s and 540 nm/s. The perfectly matched and internal mismatchedoligomer sequences are shown in Table 1, where the underlined parts aremismatched sites.

TABLE 1 Oligomer on the tip Oligomer on the tip Oligomer on theOligomer on the tip (single base internal (double base internal SIZEsubstrate (perfect matching) mismatching) mismatching) 20 5′-H₂N-5′-H₂N- 5′-H₂N- 5′-H₂N- bases CCATCGTGGTTGC CTGAGGAGCAAC CTGAGGAGCTACCTGAGGAGCTTC TCCTCAG-3′ CACGATGG-3′ CACGATGG-3′ CACGATGG-3′(SEQ ID NO: 1) (SEQ ID NO: 5) (SEQ ID NO: 9) (SEQ ID NO: 13) 30 5′-H₂N-5′-H₂N- 5′-H₂N- 5′-H₂N- bases GCTGCTATGGAG CTTCGTTCCAGGG CTTCGTTCCAGGGCTTCGTTCCAGGG ACACGCCCTGGA CGTGTCTCCATAG CGCGTCTCCATAG CTCGTCTCCATAGACGAAG-3′ CAGC-3′ CAGC-3′ CAGC-3′ (SEQ ID NO: 2) (SEQ ID NO: 6)(SEQ ID NO: 10) (SEQ ID NO: 14) 40 5′-H₂N- 5′-H₂N- 5′-H₂N- 5′-H₂N- basesTGGATCTGGGGT CGAGCACACCTT CGAGCACACCTT CGAGCACACCTT GCCATTCCGCTGTGAGACAGCGGAA GAGACAGCGTAA GAGACAGCGTCA CTCAAGGTGTGCT TGGCACCCCAGATGGCACCCCAGA TGGCACCCCAGA CG-3′ TCCA-3′ TCCA-3′ TCCA-3′ (SEQ ID NO: 3)(SEQ ID NO: 7) (SEQ ID NO: 11) (SEQ ID NO: 15) 50 5′-H₂N- 5′-H₂N-5′-H₂N- 5′-H₂N- bases GTCTGACCTGTTC TGGAGAGCAGGC TGGAGAGCAGGCTGGAGAGCAGGC CAACGACCCGTA AGGAGCGGAGTG AGGAGCGGAGTG AGGAGCGGAGTGTCACTCCGCTCCT ATACGGGTCGTT TTACGGGTCGTTG TAACGGGTCGTT GCCTGCTCTC CA-GGAACAGGTCAG GAACAGGTCAGA GGAACAGGTCAG 3′ AC-3′ C-3′ AC-3′(SEQ ID NO: 4) (SEQ ID NO: 8) (SEQ ID NO: 12) (SEQ ID NO: 16)

A solid substrate with a dendron controlled meso space maintains evendistancing among monomolecules, thus effectively widening researchapplications to drug screening, investigation of protein-proteininteraction and protein-small molecule interaction. Bio-AFM enableshighly accurate measurements while minimizing molecular damage.

The present invention maintains even spacing between biomolecules fixedon the surface of solid substrate dendron, with adjusted meso spacestructure. Unwanted steric hindrance is minimized, providing an optimalenvironment for observing interactions between monomolecules. The termbiomolecule encompasses substances such as proteins, antigens,antibodies, signaling proteins, peptides, integral membrane proteins,small molecules, steroids, glucose, DNA, RNA, and others.

Specifically, according to Examples 8 and 9, and FIGS. 11 to 15, it hasbeen demonstrated that the present invention causes uniform forcebetween PLD1-PX and Munc-18-1, which form a specific bond. The presentinvention can be applied to drug screening by measuring interactionforce using Bio-AFM. By observing the change of interaction forcebetween biomolecules in accordance to changes in the environment, causeand effect relations can be more clearly established for a wide array ofdiseases that are believed to be caused by alterations in biologicalcomposition. In addition, the dendron of the present invention allowsmeasurement of interaction force between singular biotin andstreptavidin by adjusting spacing between biotin molecules.

Dendron types that are applicable to the present invention are furtherindicated in detail in U.S. patent application Ser. No. 10/917,601 andWO 2005/026191, the contents of which are incorporated by referenceherein in their entirety.

The present invention, as shown in subsequent examples, maintains amplespace between ligand molecules by fixing a ligand onto the AFM tip withthe help of a dendron that can manipulate meso space. This minimizesunwanted steric hindrance and static interaction between ligand andreceptor, providing an optimal environment for binding. This environmentalso minimizes multiple bonding, and thus allows close observation ofligand-receptor interaction on the monomolecular level.

The present invention, by using a surface of controlled meso spacestructure when introducing an AFM tip loaded with specific ligands(which form specific bonds with cell surface receptors) maintains evenspacing between ligands and thus allows for accurate mapping of receptordistribution. When using AFM tips which meso space have not beenadjusted, however, ligand distribution becomes uneven and multiple bondswith the receptor diminishes mapping accuracy. Therefore the presentinvention presents versatile uses in studying ligand-receptorinteractions.

Cell receptors can include but are not limited to small molecules,peptides, proteins, steroid hormone receptors, carbohydrates, lipids,membrane proteins, glycoproteins, glycolipids, lectin, neurotrophinreceptors, DNA, and RNA. Any substance that exists on the cell surfaceand is capable of interaction with ligand fixed onto an AFM Tip surfacecan be used as a receptor.

Additionally, ligand types fixed onto the AFM Tip can include but arenot limited to small molecules, peptides, proteins, steroids,carbohydrates, lipids, membrane proteins, neurotrophins, antibodies,DNA, RNA, and complex compounds. In other words, any substance that canbe loaded onto an AFM Tip and is observable by interaction withreceptors can be used as ligands.

In Examples 11 to 15, and FIGS. 16 to 18 of the present invention,peptide-protein interactions, carbohydrate-glycolipid interactions,lectin-glycoprotein interactions, carbohydrate-glycoproteininteractions, and neurotrophin-neurotrophin receptor interactions weresurveyed.

The present invention is further explained in more detail with referenceto the following examples. These examples, however, should not beinterpreted as limiting the scope of the present invention in anymanner.

PREPARATION EXAMPLES

Numbering scheme is used for compounds throughout the Examples such ascompound 1, compound 2, I, II, III, IV, V and so on. It is to beunderstood however, that the compound numbering scheme is consistentwith and is confined to the particular Example section to which it isrecited. For instance, compound 1 as recited in Example 2 may notnecessarily be the same compound 1 as found in Example 3.

Example 1 Methods for Making Microarray Using Size-ControlledMacromolecule

In Example 1, designations I, II, III, IV, and V refer to variouscompounds and intermediate compounds as shown in FIG. 2.

Example 1.1 Materials

The silane coupling reagents, (3-glycidoxypropyl)methyldiethoxysilane(GPDES) and (3-aminopropyl)diethoxymethylsilane (APDES), were purchasedfrom Gelest, Inc. and all other chemicals were of reagent grade fromSigma-Aldrich. Reaction solvents for the silylation are anhydrous onesin Sure/Seal bottles from Aldrich. All washing solvents for thesubstrates are of HPLC grade from Mallinckrodt Laboratory Chemicals. TheUV grade fused silica plates (30 mm×10 mm×1.5 mm) were purchased fromCVI Laser Corporation. The polished prime Si(100) wafers (dopant,phosphorus; resistivity, 1.5-2.1 Ω·cm) were purchased from MEMCElectronic Materials, Inc. Glass slides (2.5×7.5 cm) were purchased fromCorning Co. All of the oligonucleotides were purchased from Metabion.Ultrapure water (18 M Ω/cm) was obtained from a Milli-Q purificationsystem (Millipore).

Example 1.2 Instruments

The film thickness was measured with a spectroscopic ellipsometer (J. A.Woollam Co. Model M-44). UV-vis spectra were recorded on aHewlett-Packard diodearray 8453 spectrophotometer. Tapping mode AFMexperiments were performed with a Nanoscope IIIa AFM (DigitalInstruments) equipped with an “E” type scanner.

Example 1.3 Cleaning the Substrates

Substrates such as oxidized silicon wafer, fused silica, and glassslide, were immersed into Piranha solution (conc. H₂SO₄:30% H₂O₂=7:3(v/v)) and the reaction bottle containing the solution and thesubstrates was sonicated for an hour. (Caution: Piranha solution canoxidize organic materials explosively. Avoid contact with oxidizablematerials.) The plates were washed and rinsed thoroughly with a copiousamount of deionized water after the sonication. The clean substrateswere dried in a vacuum chamber (30-40 mTorr) for the next steps.

Example 1.4 Preparing the Hydroxylated Substrates

The above clean substrates were soaked in 160 ml toluene solution with1.0 ml (3-glycidoxypropyl)methyldiethoxysilane (GPDES) for 10 h. Afterthe self-assembly, the substrates were washed with toluene briefly,placed in an oven, and heated at 110° C. for 30 min. The plates weresonicated in toluene, toluene-methanol (1:1 (v/v)), and methanol in asequential manner for 3 min at each washing step. The washed plates weredried in a vacuum chamber (30-40 mTorr). GPDES-modified substrates weresoaked in a neat ethylene glycol (EG) solution with two or three dropsof 95% sulfuric acid at 80-100° C. for 8 h. After cooling, thesubstrates were sonicated in ethanol and methanol in a sequential mannereach for 3 min. The washed plates were dried in a vacuum chamber (30-40mTorr).

Example 1.5 Preparing the Dendron-Modified Substrates

The above hydroxylated substrates were immersed into a methylenechloride solution dissolving the dendron (1.2 mM) and a coupling agent,1-[-3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) or1,3-dicyclohexylcarbodiimide (DCC) (11 mM) in the presence of4-dimethylaminopyridine (DMAP) (0.82 mM). After 3 days at roomtemperature, the plates were sonicated in methanol, water, and methanolin a sequential manner each for 3 min. The washed plates were dried in avacuum chamber (30-40 mTorr) for the next step.

Example 1.6 Preparing the NHS-Modified Substrates

The dendron-modified substrates were immersed into a methylene chloridesolution with 1.0 M trifluoroacetic acid (TFA). After 3 h, they wereagain soaked in a methylene chloride solution with 20% (v/v)diisopropylethylamine (DIPEA) for 10 min. The plates were sonicated inmethylene chloride and methanol each for 3 min. After being dried in avacuum chamber, the deprotected substrates were incubated in theacetonitrile solution with di(N-succinimidyl)carbonate (DSC) (25 mM) andDIPEA (1.0 mM). After 4 h reaction under nitrogen atmosphere, the plateswere placed in a stirred dimethylformamide solution for 30 min andwashed briefly with methanol. The washed plates were dried in a vacuumchamber (30-40 mTorr) for the next step.

Example 1.7 Arraying Oligonucleotides on the NHS-Modified Substrates

Probe oligonucleotides in 50 mM NaHCO3 buffer (pH 8.5) were spotted sideby side in a 4 by 4 format on the NHS-modified substrate. The microarraywas incubated in a humidity chamber (80% humidity) for 12 h to give theamine-tethered DNA sufficient reaction time. Slides were then stirred ina hybridization buffer solution (2×SSPE buffer (pH 7.4) containing 7.0mM sodium dodecylsulfate) at 37° C. for 1 h and in boiling water for 5min to remove non-specifically bound oligonucleotides. Finally, theDNA-functionalized microarray was dried under a stream of nitrogen forthe next step. For a fair comparison, different kinds of probes werespotted in a single plate.

Example 1.8 Hybridization

Hybridization was performed in the hybridization buffer solutioncontaining a target oligonucleotide (1.0 nM) tagged with a Cy3fluorescent dye at 50° C. for 1 h using a GeneTAC™ HybStation (GenomicSolutions, Inc.). The microarray was rinsed with the hybridizationbuffer solution in order to remove excess target oligonucleotide anddried with nitrogen. The fluorescence signal on each spot was measuredwith a ScanArray Lite (GSI Lumonics) and analyzed by Imagene 4.0(Biodiscovery).

Example 1.9 Synthesis of the Dendron Example 1.9.1 Preparation of9-anthrylmethyl N-(3-carboxylpropyl)carbamate (I)—Compound I

4-Aminobutyric acid (0.50 g, 4.8 mmol, 1.0 equiv) and triethylamine(TEA) (1.0 ml, 7.3 mmol, 1.5 equiv) were dissolved inN,N-dimethylformamide (DMF) and stirred at 50° C. 9-Anthrylmethylρ-nitrophenyl carbonate (1.81 g, 4.8 mmol, 1.0 equiv) was slowly addedwhile stirring. After stirring at 50° C. for 2 h, the solution wasevaporated to dryness, and the solution was basified with 0.50 N sodiumhydroxide (NaOH) solution. The aqueous solution was washed with ethylacetate (EA), stirred in an ice bath and acidified with dilutehydrochloric acid (HCI). After the product was extracted with EA, theorganic solution was dried with anhydrous MgSO₄, filtered andevaporated. The total weight of the resulting yellow powder was 1.06 gand the yield was 65%.

¹H NMR (CDCl₃)

δ 11.00-9.00 (br, CH₂COOH, 1H), 8.41 (s, C₁₄H₉CH₂, 1H), 8.31 (d,C₁₄H₉CH₂, 2H), 7.97 (d, C₁₄H₉CH₂, 2H), 7.51 (t, C₁₄H₉CH₂, 2H), 7.46 (t,C₁₄H₉CH₂, 2H), 6.08 (s, C₁₄H₉CH₂O, 2H), 5.01 (t, OCONHCH₂, 1H), 3.23 (q,NHCH₂CH₂, 2H), 2.34 (t, CH₂CH₂COOH, 2H), 1.77 (m, CH₂CH₂CH₂, 2H).

¹³C NMR (CDCl3)

δ 178.5 (CH₂COOH), 157.9 (OCONH), 132.1 (C₁₄H₉CH₂), 131.7 (C₁₄H₉CH₂),129.7 (C₁₄H₉CH₂), 129.7 (C₁₄H₉CH₂), 127.3 (C₁₄H₉CH₂), 126.8 (C₁₄H₉CH₂),125.8 (C₁₄H₉CH₂), 124.6 (C₁₄H₉CH₂), 60.2 (C₁₄H₉CH₂), 41.0 (NHCH₂CH₂),31.7 (CH₂CH₂COOH), 25.6 (CH₂CH₂CH₂).

Example 1.9.2 Preparation of 9-anthrylmethylN-{[(tris{[2-(methoxycarbonyl)ethoxy]methyl}methyl)amino]carbonyl}propylcarbonate(II)—Compound II

9-Anthrylmethyl N-(3-carboxylpropyl)carbamate (0.65 g, 1.93 mmol, 1.5equiv), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride(EDC) (0.37 g, 1.93 mmol, 1.5 equiv), and 1-hydroxybenzotriazole hydrate(HOBT) (0.261 g, 1.93 mmol, 1.5 equiv) were dissolved in acetonitrileand stirred at room temperature.Tris{[(methoxycarbonyl)ethoxy]methyl}aminomethane (0.49 g, 1.29 mmol,1.0 equiv) dissolved in acetonitrile was added with stirring, Afterstirring at room temperature for 12 h, the acetonitrile was evaporated.The crude product was dissolved in EA and washed with 1.0 N HCl andsaturated sodium bicarbonate solution. After being dried with anhydrousMgSO₄, filtered, and evaporated, the crude product was loaded in acolumn packed with silica gel. Purification by column chromatography(eluent:ethyl acetate:hexane=5:1 (v/v)) resulted in a viscous yellowliquid. The total weight of the yellow liquid was 0.67 g, and the yieldwas 74%.

¹H NMR (CDCl₃)

δ 8.43 (s, C₁₄H₉CH₂, 1H), 8.36 (d, C₁₄H₉CH₂, 2H), 7.99 (d, C₁₄H₉CH₂,2H), 7.53 (t, C₁₄H₉CH₂, 2H), 7.47 (t, C₁₄H₉CH₂, 2H), 6.15 (s, CONHC,1H), 6.08 (s, C₁₄H₉CH₂O, 2H), 5.44 (t, OCONHCH₂, 1H), 3.63-3.55 (m,CH₂OCH₂CH₂COOCH₃, 21H), 3.27 (q, NHCH₂CH₂, 2H), 2.46 (t, CH₂CH₂COOCH₃,6H), 2.46 (t, CH₂CH₂CONH, 2H), 1.81 (m, CH₂CH₂CH₂, 2H).

¹³C NMR (CDCl₃)

δ173.2 (CH₂CONH), 172.7 (CH₂COOCH₃), 157.4 (OCONH), 132.9 (C₁₄H₉CH₂),131.5 (C₁₄H₉CH₂), 129.5 (C₁₄H₉CH₂), 129.4 (C₁₄H₉CH₂}, 127.5 (C₁₄H₉CH₂),127.0 (C₁₄H₉CH₂), 125.6 (C₁₄H₉CH₂), 124.7 (C₁₄H₉CH₂), 69.6 (NHCCH₂O),67.2 (C₁₄H₉CH₂), 60.1 (OCH₂CH₂), 59.4 (NHCCH₂), 52.1 (OCH₃), 40.8(NHCH₂CH₂), 35.1 (OCH₂CH₂), 34.7 (CH₂CH₂CONH), 26.3 (CH₂CH₂CH₂).

Anal. Calcd for C₃₆H₄₆N₂O₁₂.0.5 H₂O: C, 61.18; H, 6.65; N, 4.03. Found:C, 61.09; H, 6.69; N, 3.96.

Example 1.9.3 Preparation of 9-anthrylmethylN-[({tris[(2-carboxyethoxy)methyl]methyl}amino)carbonyl]propylcarbamate(III)—Compound III

9-AnthrylmethylN-{[(tris{[2-(methoxycarbonyl)ethoxy]methyl}methyl)amino]carbonyl}propyl-carbonate(0.67 g, 0.93 mmol) was dissolved in acetone (30 ml) and 0.20 N NaOH (30ml, 6 mmol). After being stirred at room temperature for 1 d, theacetone was evaporated. The aqueous solution was washed with EA, stirredin an ice bath and acidified with dilute HCl. After the product wasextracted with EA, the organic solution was dried with anhydrous MgSO₄,filtered and evaporated. Solidification in acetone and ether solution at−20° C. resulted in a yellow powder. The total weight of the final paleyellow powder was 0.54 g with a yield of 88%.

¹H NMR(CDCl₃)

δ 11.00-9.00 (br, CH₂COOH, 3H}, 8.61 (s, C₁₄H₉CH₂, 1H}, 8.47 (d,C₁₄H₉CH₂, 2H), 8.11 (d, C₁₄H₉CH₂, 2H), 7.60 (t, C₁₄H₉CH₂, 2H}, C₁₄H₉CH₂,2H), 6.63 (s, CONHC, 1H), 6.36 (t, OCONHCH₂, 1H), 6.12 (s, C₁₄H₉CH₂O,2H). 3.40-363 (m, CH₂OCH₂CH₂COOH, 12H), 3.20 (q, NHCH₂CH₂, 2H), 2.52 (t,CH₂CH₂COOH, 6H), 2.17 (t, CH₂CH₂CONH, 2H), 1.75 (m, CH₂CH₂CH₂, 2H).

¹³C NMR (CDCl₃)

δ 172.2 (CH₂COOH), 172.0 (CH₂CONH), 156.7 (OCONH), 131.2 (C₁₄H₉CH₂),130.7 (C₁₄H₉CH₂), 128.6 (C₁₄H₉CH₂), 128.4 (C₁₄H₉CH₂), 127.3 (C₁₄H₉CH₂),126.2 (C₁₄H₉CH₂), 124.8 (C₁₄H₉CH₂), 124.0 (C₁₄H₉CH₂), 68.6 (NHCCH₂O),66.5 (C₁₄H₉CH₂), 59.5 (OCH₂CH₂), 58.0 (NHCCH₂), 40.0 (NHCH₂CH₂), 34.0(OCH₂CH₂), 33.5 (CH₂CH₂CONH), 25.8 (CH₂CH₂CH₂).

Anal. Calcd for C₃₃H₄₀N₂O₁₂.1.5 H₂O: C, 57.97; H, 6.34; N, 4.10. Found:C, 57.89; H, 6.21; N, 4.09.

Example 1.9.4 Preparation of 9-anthrylmethylN-[({tris[(2-{[(tris{[2-(methoxycarbonyl)ethoxy]methyl}(methyl)amino]carbonyl}ethoxy)methyl]methyl}amino)carbonyl]propylcarbamate(IV)—Compound IV

9-AnthrylmethylN-[({tris[(2-carboxyethoxy)methyl]methyl}amino)carbonyl]propylcarbamate(0.54 g, 0.82 mmol, 1.0 equiv), EDC (0.55 g, 2.87 mmol, 3.5 equiv), andHOBT (0.39 g, 2.89 mmol, 3.5 equiv) were dissolved in acetonitrile andstirred at room temperature.Tris{[(methoxycarbonyl)ethoxy]methyl}aminomethane (0.96 g, 2.53 mmol,3.1 equiv) dissolved in acetonitrile was added with stirring. Afterstirring at room temperature for 36 h, the acetonitrile was evaporated.The crude product was dissolved in EA and washed with 1.0 N HCl andsaturated sodium bicarbonate solution. After drying with anhydrousMgSO₄, filtered, and evaporated, the crude product was loaded in acolumn packed with silica gel. Column purification (eluent:ethylacetate:methanol=20:1 (v/v)) resulted in a viscous yellow liquid. Thetotal weight of the yellow liquid was 1.26 g with an 88% yield.

¹H NMR (CDCl₃)

δ 8.47 (s, C₁₄H₉CH₂, 1H), 8.39 (d, C₁₄H₉CH₂, 2H), 8.02 (d, C₁₄H₉CH₂,2H), 7.53 (t, C₁₄H₉CH₂, 2H), 7.47 (t, C₁₄H₉CH₂, 2H), 6.60 (s,CH₂CH₂CH₂CONHC, 1H), 6.13 (s, OCH₂CH₂CONHC, 3H), 6.11 (s, C₁₄H₉CH₂O,2H), 5.79 (t, OCONHCH₂, 1H), 3.65-3.60 (m, CH₂OCH₂CH₂CONH,CH₂OCH₂CH₂COOCH₃, 75H), 3.29 (q, NHCH₂CH₂, 2H), 2.50 (t, CH₂CH₂COOCH₃,18H), 2.36 (t, OCH₂CH₂CONH, 6H), 2.27 (t, CH₂CH₂CH₂CONH, 2H), 1.85 (m,CH₂CH₂CH₂, 2H).

¹³C NMR (CDCl₃)

δ 173.3 (OCH₂CH₂CONH), 172.5 (CH₂CH₂CH₂CONH), 171.6 (CH₂COOCH₃), 157.2(OCONH), 131.8 (C₁₄H₉CH₂), 131.5 (C₁₄H₉CH₂), 129.4 (C₁₄H₉CH₂), 129.3(C₁₄H₉CH₂), 127.6 (C₁₄H₉CH₂), 127.0 (C₁₄H₉CH₂), 125.6 (C₁₄H₉CH₂), 124.7(C₁₄H₉CH₂), 69.5 (NHCCH₂OCH₂CH₂COOCH₃), 67.9 (NHCCH₂OCH₂CH₂CONH), 67.2(C₁₄H₉CH₂), 60.3 (OCH₂CH₂CONH), 60.2 (OCH₂CH₂COOCH₃), 59.2(NHCCH₂OCH₂CH₂COOCH₃, NHCCH₂OCH₂CH₂CONH), 52.1 (OCH₃), 41.0 (NHCH₂CH₂),37.6 (OCH₂CH₂CONH), 35.1 (OCH₂CH₂COOCH₃), 34.7 (CH₂CH₂CH₂CONH), 26.3(CH₂CH₂CH₂).

Anal. Calcd for C₈₁H₁₂₁N₅O₃₆.H₂O: C, 55.31; H, 7.05; N, 3.98. Found: C,55.05; H, 7.08; N, 4.04.

MALDI-TOF-MS: 1763.2 (MNa+), 1779.2 (MK+).

Example 1.9.5 Preparation of 9-anthrylmethylN-({[tris({2-[({tris[(2-carboxyethoxy)methyl]methyl}amino)carbonyl]ethoxy}methyl)methyl]amino}carbonyl)propylcarbamate(V—Compound V

9-Anthrylmethyl N-[({tris[(2-{[(tris{[2-(methoxycarbonyl)ethoxy]methyl}methyl)amino]carbonyl}ethoxy)methyl]methyl}amino)carbonyl]propylcarbamate(0.60 g, 0.34 mmol) was dissolved in acetone (30 ml) and 0.20 N NaOH (30ml). After stirring at room temperature for 1 d, the acetone wasevaporated. The aqueous solution was washed with EA, stirred in an icebath and acidified with dilute HCI. After the product was extracted withEA, the organic solution was dried with anhydrous MgSO₄, filtered andevaporated. The total weight of the final yellow powder was 0.37 g andthe yield was 68%.

¹H NMR (DMSO)

δ 13.00-11.00 (br, CH₂COOH, 9H), 8.66 (s, C₁₄H₉CH2, 1H), 8.42 (d,C₁₄H₉CH₂, 2H), 8.13 (d, C₁₄H₉CH2, 2H), 7.62 (t, C₁₄H₉CH₂, 2H), 7.54 (t,C₁₄H₉CH₂, 2H), 7.12 (t, OCONHCH₂, 1H), 7.10 (s, OCH₂CH₂CONHC, 3H), 7.06(s, CH₂CH₂CH₂CONHC, 1H), 6.06 (s, C₁₄H₉CH₂O, 2H), 3.57-3.55 (m,CH₂OCH₂CH₂CONH, CH₂OCH₂CH₂COOH, 48H), 3.02 (q, NHCH₂CH₂, 2H), 2.42 (t,CH₂CH₂COOH, 18H), 2.32 (t, OCH₂CH₂CONH, 6H), 2.11 (t, CH₂CH₂CH₂CONH,2H), 1.60 (m, CH₂CH₂CH₂, 2H).

¹³C NMR (DMSO)

δ 172.8 (CH₂COOH), 172.2 (CH₂CH₂CH₂CONH), 170.5 (OCH₂CH₂CONH), 156.5(OCONH), 131.0 (C₁₄H₉CH₂), 130.6 (C₁₄H₉CH₂), 129.0 (C₁₄H₉CH₂), 128.7(C₁₄H₉CH₂), 127.6 (C₁₄H₉CH₂), 126.7 (C₁₄H₉CH₂), 125.4 (C₁₄H₉CH₂), 124.3(C₁₄H₉CH₂), 68.3 (NHCCH₂OCH₂CH₂COOH), 67.4 (NHCCH₂OCH₂CH₂CONH), 66.8(C₁₄H₉CH₂), 59.8 (OCH₂CH₂COOH), 59.6 (OCH₂CH₂CONH), 57.9(NHCCH₂OCH₂CH₂CONH), 55.9 (NHCCH₂OCH₂CH₂COOH), 36.4 (NHCH₂CH₂), 34.6(OCH₂CH₂COOH), 30.8 (OCH₂CH₂CONH), 29.7 (CH₂CH₂CH₂CONH), 25.9(CH₂CH₂CH₂).

Preparation Example 2 Methods of Producing Alternative Starting MaterialDendron Macromolecule—Fmoc-Spacer-[9]-Acid

In Example 2, various indicated compounds are referred to as compound 1,2 and so forth.

First, a spacer, 6-azidohexylamine (1) from 1,6-dibromohexane wassynthesized according to Lee, J. W.; Jun, S. I.; Kim, K. TetrahedronLett., 2001, 42, 2709.

This spacer was attached to repeating unit (2) through unsymmetric ureaformation and made N₃-spacer-[3]ester (3). The repeating unit wassynthesized by condensation of TRIS with tert-butyl acrylate, which hadbeen reported in Cardona, C. M.; Gawley, R. E. J. Org. Chem. 2002, 67,141.

This triester was transformed to N₃-spacer-[3]acid (4) throughhydrolysis and coupled with triester (2) under peptide couplingconditions, which led to N₃-spacer-[9]ester. After reduction of azide toamine and protection of amine with Fmoc group, hydrolysis of nonaesterafforded Fmoc-spacer-[9]acid (5).

N-(6-Azidohexyl)-N′-tris{[2-(tert-butoxycarbonyl)ethoxy]methyl}-methylurea (3)

Triphosgene (1.3 g, 4.3 mmol) was dissolved in anhydrous CH₂Cl₂ (20 mL).A mixture of 6-azidohexylamine (1) (1.6 g, 12 mmol) andN,N-diisopropylethylamine (DIEA, 2.4 mL, 13.8 mmol) in anhydrous CH₂Cl₂(35 mL) was added dropwise to the stirred solution of triphosgene over aperiod of 7 h using a syringe pump. After further stirring for 2 h, asolution of (2) (6.4 g, 13 mmol) and DIEA (2.7 mL, 15.2 mmol) inanhydrous CH₂Cl₂ (20 mL) was added. The reaction mixture was stirred for4 h at room temperature under nitrogen, and washed with 0.5 M HCl andbrine. The organic layer was then dried over anhydrous MgSO₄, and thesolvent was removed by evacuation. Purification with columnchromatography (silica, 1:1 EtOAc/hexane) yielded colorless oil (3.0 g,40%).

¹H NMR (CDCl₃, 300 MHz): δ 1.45 (s, (CH₃)₃C, 27H); 1.36-1.58 (m,CH₂CH₂CH₂CH₂, 8H); 2.46 (t, CH₂CH₂O, J=6.4 Hz, 6H), 3.13 (m, CONHCH₂,2H), 3.26 (t, N₃CH₂, J=6.9 Hz, 2H), 3.64-3.76 (m, CCH₂O and CH₂CH₂O,12H); 5.00 (t, CH₂NHCO, J=6.7 Hz, 1H), 5.29 (s, CONHC, 1H).

¹³C NMR (CDCl₃, 75 MHz): δ 26.52, 26.54, 28.81, 30.26 (CH₂CH₂CH₂CH₂);28.14 ((CH₃)₃C); 36.20 (CH₂CH₂O); 39.86 (CONHCH₂); 51.40 (N₃CH₂); 58.81(CCH₂O); 67.16 (CH₂CH₂O); 69.23 (CCH₂O); 80.58 ((CH₃)₃C); 157.96(NHCONH); 171.26 (COOt-Bu).

FAB-MS: 674.26 (M⁺).

N-(6-Azidohexyl)-N′-tris {[2-carboxyethoxy]methyl}methylurea (4)

N₃-spacer-[3]ester (3) (0.36 g, 0.56 mmol) was stirred in 6.6 mL of 96%formic acid for 24 h. The formic acid was then removed at reducedpressure at 50° C. to produce colorless oil in a quantitative yield.

¹H NMR (CD₃COCD₃, 300 MHz): δ 1.34-1.60 (m, CH₂CH₂CH₂CH₂, 8H); 2.53 (t,CH₂CH₂O, J=6.4 Hz, 6H), 3.07 (t, CONHCH₂, J=6.9 Hz, 2H), 3.32 (t, N₃CH₂,J=6.9 Hz, 2H), 3.67-3.73 (m, CCH₂O and CH₂CH₂O, 12H).

¹³C NMR (CD₃COCD₃, 75 MHz): δ 27.21, 29.54, 31.02 (CH₂CH₂CH₂CH₂); 35.42(CH₂CH₂O); 40.27 (CONHCH₂); 52.00 (N₃CH₂); 59.74 (CCH₂O); 67.85(CH₂CH₂O); 70.96 (CCH₂O); 158.96 (NHCONH); 173.42 (COOH).

FAB-MS: 506.19 (MH⁺).

N-(6-Azidohexyl)-N′-tris[(2-{[(tris{[2-(tert-butoxycarbonyl)ethoxy]-methyl}methyl)amino]carbonyl}ethoxy)methyl]methylurea(4.1)

The HOBt (0.20 g, 1.5 mmol), DIEA (0.30 mL, 1.8 mmol), and EDC (0.33 g,1.8 mmol) were added to (4) (0.25 g, 0.50 mmol) in 5.0 mL of dryacetonitrile. Then, the amine (2) (1.14 g, 2.3 mmol) dissolved in 2.5 mLof dry acetonitrile was added, and the reaction mixture was stirredunder N₂ for 48 h. After removal of the solvent at reduced pressure, theresidue was dissolved in MC and washed with 0.5 M HCl and brine. Theorganic layer was then dried over MgSO₄, the solvent was removed invacuo, and column chromatography (SiO2, 2:1 EtOAc/hexane) yielded acolorless oil (0.67 g, 70%).

¹H NMR (CDCl₃, 300 MHz): δ 1.45 (s, (CH₃)₃C, 81H); 1.36-1.58 (m,CH₂CH₂CH₂CH₂, 8H); 2.40-2.47 (m, CH₂CH₂O gen. 1 & 2, 24H), 3.13 (m,CONHCH₂, 2H), 3.26 (t, N₃CH₂, 6.9 Hz, 2H), 3.62-3.69 (m, CCH₂O gen. 1 &2, CH₂CH₂O gen. 1 & 2, 48H); 5.36 (t, CH₂NHCO, J=6.7 Hz, 1H), 5.68 (br,CONHC, 1H), 6.28 (br, amide NH, 3H).

¹³C NMR (CDCl₃, 75 MHz): δ 26.59, 26.69, 28.91, 30.54 (CH₂CH₂CH₂CH₂);28.22 ((CH₃)₃C); 36.20 (CH₂CH₂O gen. 2); 37.43 (CH₂CH₂O gen. 1); 39.81(CONHCH₂); 51.47 (N₃CH₂); 58.93 (CCH₂O gen. 1); 59.89 (CCH₂O gen. 2);67.15 (CH₂CH₂O gen. 2); 67.68 (CH₂CH₂O gen. 1); 69.23 (CCH₂O gen. 2);70.12 (CCH₂O gen. 1); 80.57 ((CH₃)₃C); 158.25 (NHCONH); 171.01 (COOt-Bu)171.41 (CONH amides).

MALDI-MS: 1989.8 (MNa⁺), 2005.8 (MK⁺).

N-(6-Aminohexyl)-N′-tris[(2-{[(tris{[2-(tert-butoxycarbonyl)ethoxy]-methyl}methyl)amino]carbonyl}ethoxy)methyl]methylurea(4.2)

Nona-tert-butyl ester (4.1) (0.37 g, 0.20 mmol) was stirred with 10%Pd/C (37.0 mg) in ethanol (20.0 mL) under H₂ at room temperature for 12h. After checking completion of the reaction with TLC, the mixture wasfiltered with a 0.2 μm Millipore filter. After the filter paper wasrinsed with CH₂Cl₂, the combined solvent was removed in vacuo, andcolorless oil was recovered.

N-{6-(9-fluorenylmethoxycarbonyl)aminohexyl}-N′-tris[(2-{[(tris{[2-(tert-butoxycarbonyl)ethoxy]methyl}methyl)amino]carbonyl}ethoxy)methyl]methylurea(4.3)

The amine (4.2) (0.33 g, 0.17 mmol) and DIEA (33 μL, 0.19 mmol) weredissolved in 5.0 mL of CH₂Cl₂, and stirred for 30 min under nitrogenatmosphere. 9-Fluorenylmethyl chloroformate (48 mg, 0.19 mmol) in 2.0 mLof CH₂Cl₂ was added, and the reaction mixture was stirred for 3 h atroom temperature. The solvent was removed under reduced pressure andwashed with 0.5 M HCl and brine. The residue was purified with columnchromatography (silica, EtOAc) to yield colorless oil (0.18 g, 64%).

¹H NMR (CDCl₃, 300 MHz): δ 1.45 (s, (CH₃)₃C, 81H); 1.23-1.58 (m,CH₂CH₂CH₂CH₂, 8H); 2.37-2.47 (m, CH₂CH₂O gen. 1 & 2, 24H); 3.10-3.22 (m,CONHCH₂, 4H); 3.62-3.70 (m, CCH₂O gen. 1 & 2, CH₂CH₂O gen. 1 & 2, 48H);4.22 (t, CH(fluorenyl)-CH₂, J=7.1 Hz, 1H); 4.36 (d, fluorenyl-CH₂, J=7.1Hz, 2H); 5.27-5.35 (m, CH₂NHCO, 2H); 5.67 (br, CONHC, 1H); 6.25 (br,amide, 3H); 7.28-7.77 (fluorenyl, 8H).

¹³C NMR (CDCl₃, 75 MHz): δ 26.85, 27.02, 30.27, 30.88 (CH₂CH₂CH₂CH₂);28.49 ((CH₃)₃C); 36.48 (CH₂CH₂O gen. 2); 37.73 (CH₂CH₂O gen. 1); 40.03,41.34 (CONHCH₂); 47.68 (CH(fluorenyl)-CH₂); 59.22 (CCH₂O gen. 1); 60.16(CCH₂O gen. 2); 66.87 (fluorenyl-CH₂); 67.43 (CH₂CH₂O gen. 2); 67.98(CH₂CH₂O gen. 1); 69.52 (CCH₂O gen. 2); 70.42 (CCH₂O gen. 1); 80.84((CH₃)₃C); 120.28, 125.52, 127.38, 127.98, 141.65, 144.48 (fluorenyl);156.88 (OCONH); 158.52 (NHCONH); 171.27 (COOt-Bu) 171.65 (amide CONH).

MALDI-MS: 2186.8 (MNa⁺), 2002.8 (MK⁺).

N-{6-(9-fluorenylmethoxycarbonyl)aminohexyl}-N′-tris[(2-{[(tris{[2-carboxyethoxy]methyl}methyl)amino]carbonyl}ethoxy)methyl]-methylurea(5)

Nona-tert-butyl ester having a protecting group (4.3) (0.12 g, 72 mmol)was stirred in 10 mL of 96% formic acid for 18 h. The formic acid wasthen removed at reduced pressure at 50° C. to produce colorless oil in aquantitative yield.

¹H NMR (CD₃COCD₃, 300 MHz): δ 1.23-1.51 (m, CH₂CH₂CH₂CH₂, 8H); 2.44-2.58(m, CH₂CH₂O gen. 1 & 2, 24H); 3.15-3.18 (m, CONHCH₂, 4H); 3.61-3.75 (m,CCH₂O gen. 1 & 2, CH₂CH₂O gen. 1 & 2, 48H); 4.23 (t, CH(fluorenyl)-CH₂,J=7.0 Hz, 1H); 4.35 (d, fluorenyl-CH₂, J=7.0 Hz, 2H); 5.85, 6.09 (br,CH₂NHCO, 2H); 6.57 (br, CONHC, 1H); 6.88 (br, amide NH, 3H); 7.31-7.88(fluorenyl, 8H).

¹³C NMR (CD₃COCD₃, 75 MHz): δ 27.21, 27.33, 30.69, 30.98 (CH₂CH₂CH₂CH₂);35.31 (CH₂CH₂O gen. 2); 37.83 (CH₂CH₂O gen. 1); 40.56, 41.54 (CONHCH₂);48.10 (CH(fluorenyl)-CH₂); 59.93 (CCH₂O gen. 1); 61.10 (CCH₂O gen. 2);66.86 (fluorenyl-CH₂); 67.81 (CH₂CH₂O gen. 2); 68.37 (CH₂CH₂O gen. 1);69.80 (CCH₂O gen. 2); 70.83 (CCH₂O gen. 1); 120.84, 126.13, 127.98,128.56, 142.10, 145.16 (fluorenyl); 157.50 (OCONH); 159.82 (NHCONH);173.20 (amide CONH); 173.93 (COOH).

Example 3 Additional Dendron Compounds

It is to be noted that while a particular protecting group may be shownwith a macromolecule, the compounds are not limited to the specificprotecting groups shown. Moreover, while various chains and spacers aredepicted indicating an exact molecular structure, modifications arepossible according to accepted chemical modification methods to achievethe function of a density controlled, preferably low density, array on asubstrate surface. As a point of reference for the short-handdescription of the compounds, the left most letter(s) indicates theprotecting group; the numeral in brackets indicates the number ofbranched termini; and the right most chemical entity indicates thechemistry on the branched termini. For example, “A-[27]-acid” indicatesanthrylmethyl protecting group; 27 termini, and acid groups at thetermini.

Example 3.1 Preparation Methods 1. A-[3]-OEt (3)

Compound 1 reacted with NaC(CO₂Et)₃ 2 in C₆H₆/DMF at 80° C.

2. A-[3]-OMe (5)

A-[3]-OEt 3 was reduced with LiAlH₄ or LiBH₄ in ether, reacted withchloroacetic acid in the presence of t-BuOK/t-BUOH, and esterified withMeOH.

3. A-[3]-OTs (7)

Reduction of A-[3]-OMe 5 with LiAlH₄ in ether yields triol compound 6,which is tosylated to compound 7.

4. A-[9]-OEt (8)

A-[3]-OTs 7 was treated with NaC(CO₂Et)₃ in C₆H₆-DMF to afford thedesired nonaester (compound 8)

5. A-[27]-OH (9)

A-[9]-OEt 8 was treated with tris(hydroxymethyl)aminomethane and K₂CO₃in DMSO at 70° C.

Example 3.2 1. Boc-[2]-OMe (3)

Compound 1 was reacted with methyl acrylate 2 in methanol solvent attemperature below 50° C. Excess reagents and solvent were removed underhigh vacuum at temperature below 55° C.

2. Boc-[4]-NH₂ (5)

Boc-[2]-OMe 3 was reacted with large excesses of ethylenediamine (EDA) 4in methanol solvent at temperature below 50° C. Excess reagents andsolvent were removed under high vacuum at temperature below 55° C.

3. Boc-[8]-OMe (6)

Boc-[4]-NH₂ 5 was reacted with methyl acrylate 2 in methanol solvent attemperature below 50° C. Excess reagents and solvent were removed underhigh vacuum at temperature below 55° C.

Example 3.3 1. Boc-[2]-OH (3)

Compound 1, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride (EDC), and 1-hydroxybenzotriazole hydrate (HOBT) weredissolved in acetonitrile and stirred at room temperature. L-glutamicacid-diethyl ester (H₂NCH(CO₂Et)CH₂CH₂CO₂Et) dissolved in acetonitrilewas added with stirring, After stirring at room temperature for 12 h,the acetonitrile was evaporated. The crude product was dissolved in EAand washed with 1.0 N HCl and saturated sodium bicarbonate solution.After being dried with anhydrous MgSO₄, filtered, and evaporated, thecrude product was loaded in a column packed with silica gel.Purification by column chromatography (eluent:ethyl acetate:haxane)resulted in a viscous yellow liquid.

Compound 2 was hydrolyzed by NaOH solution. After being stirred at roomtemperature for 1 d, the organic liquid was evaporated. The aqueoussolution was washed with EA, stirred in an ice bath and acidified withdilute HCl. After the product was extracted with EA, the organicsolution was dried with anhydrous MgSO₄, filtered and evaporated.

2. Boc-[4]-OH (3)

Compound 3, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride (EDC), and 1-hydroxybenzotriazole hydrate (HOBT) weredissolved in acetonitrile and stirred at room temperature. L-glutamicacid-diethyl ester (H₂NCH(CO₂Et)CH₂CH₂CO₂Et) dissolved in acetonitrilewas added with stirring, After stirring at room temperature for 12 h,the acetonitrile was evaporated. The crude product was dissolved in EAand washed with 1.0 N HCl and saturated sodium bicarbonate solution.After being dried with anhydrous MgSO₄, filtered, and evaporated, thecrude product was loaded in a column packed with silica gel.Purification by column chromatography (eluent:ethyl acetate:haxane)resulted in a viscous yellow liquid.

Compound 4 was hydrolyzed by NaOH solution. After being stirred at roomtemperature for 1 d, the organic liquid was evaporated. The aqueoussolution was washed with EA, stirred in an ice bath and acidified withdilute HCl. After the product was extracted with EA, the organicsolution was dried with anhydrous MgSO₄, filtered and evaporated.

3. Boc-[8]-OH (3)

Compound 5, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride (EDC), and 1-hydroxybenzotriazole hydrate (HOBT) weredissolved in acetonitrile and stirred at room temperature. L-glutamicacid-diethyl ester (H₂NCH(CO₂Et)CH₂CH₂CO₂Et) dissolved in acetonitrilewas added with stirring, After stirring at room temperature for 12 h,the acetonitrile was evaporated. The crude product was dissolved in EAand washed with 1.0 N HCl and saturated sodium bicarbonate solution.After being dried with anhydrous MgSO₄, filtered, and evaporated, thecrude product was loaded in a column packed with silica gel.Purification by column chromatography (eluent:ethyl acetate:haxane)resulted in a viscous yellow liquid.

Compound 6 was hydrolyzed by NaOH solution. After being stirred at roomtemperature for 1 d, the organic liquid was evaporated. The aqueoussolution was washed with EA, stirred in an ice bath and acidified withdilute HCl. After the product was extracted with EA, the organicsolution was dried with anhydrous MgSO₄, filtered and evaporated.

Example 3.4 1. Boc-[2]-CN (3)

Compound 1 was dissolved at room temp. in acrylonitrile. Glacial aceticacid was added and the solution is heated under reflux for 24 h. Excessacrylonitrile was distilled off under vacuum, the residue was extractedwith chloroform, and added to concentrated ammonia solution. The organicphase was separated, washed with water, and dried with sodium sulfate.

2. Boc-[2]-NH₂ (4)

Boc-[2]-CN 3 was dissolved in methanol and cobalt(II) chloridehexahydrate was added. Sodium borohydride was added in portions. Theresultant mixture was stirred for 2 h at room temp. and then cautiouslyacidified with concentrated hydrochloric acid. The solvent was removedunder vacuum and concentrated. The organic phase was separated, washedwith water, and dried with sodium sulfate.

3. Boc-[4]-CN (5)

Boc-[2]-NH₂ 4 was dissolved at room temp. in acrylonitrile. Glacialacetic acid was added and the solution is heated under reflux for 24 h.Excess acrylonitrile was distilled off under vacuum, the residue wasextracted with chloroform, and added to concentrated ammonia solution.The organic phase was separated, washed with water, and dried withsodium sulfate.

4. Boc-[4]-NH₂ (6)

Boc-[4]-CN 5 was dissolved in methanol and cobalt(II) chloridehexahydrate was added. Sodium borohydride was added in portions. Theresultant mixture was stirred for 2 h at room temp. and then cautiouslyacidified with concentrated hydrochloric acid. The solvent was removedunder vacuum and concentrated. The organic phase was separated, washedwith water, and dried with sodium sulfate.

5. Boc-[8]-CN (7)

Boc-[4]-NH₂ 6 was dissolved at room temp. in acrylonitrile. Glacialacetic acid was added and the solution is heated under reflux for 24 h.Excess acrylonitrile was distilled off under vacuum, the residue wasextracted with chloroform, and added to concentrated ammonia solution.The organic phase was separated, washed with water, and dried withsodium sulfate.

6. Boc-[8]-NH₂ (8)

Boc-[8]-CN 7 was dissolved in methanol and cobalt(II) chloridehexahydrate was added. Sodium borohydride was added in portions. Theresultant mixture was stirred for 2 h at room temp. and then cautiouslyacidified with concentrated hydrochloric acid. The solvent was removedunder vacuum and concentrated. The organic phase was separated, washedwith water, and dried with sodium sulfate.

7. Boc-[16]-CN (9)

Boc-[8]-NH₂ 8 was dissolved at room temp. in acrylonitrile. Glacialacetic acid was added and the solution is heated under reflux for 24 h.Excess acrylonitrile was distilled off under vacuum, the residue wasextracted with chloroform, and added to concentrated ammonia solution.The organic phase was separated, washed with water, and dried withsodium sulfate.

7. Boc-[16]-NH₂ (10)

Boc-[16]-CN 9 was dissolved in methanol and cobalt(II) chloridehexahydrate was added. Sodium borohydride was added in portions. Theresultant mixture was stirred for 2 h at room temp. and then cautiouslyacidified with concentrated hydrochloric acid. The solvent was removedunder vacuum and concentrated. The organic phase was separated, washedwith water, and dried with sodium sulfate.

Example 3.5 1. A-[3]-Alkene (3)

A-[1]-SiCl₃ 1 was refluxed with 10% excess of allylmagnesium bromide indiethyl ether for 4 h, and cooled to 0° C. and hydrolyzed with 10%aqueous NH₄Cl. The organic layer was washed with water, dried MgSO₄ andconcentrated.

2. A-[3]-SiCl₃ (4)

A mixture of A-[3]-Alkene 3, HSiCl₃, and a common platinum-basedhydrosilylation catalyst, e.g. H2PtCl6 in propan-2-ol (Speier'scatalyst) or platinum divinylsiloxane complecx (Karstedt's catalyst),was stirred for 24 h at room temp. When the reaction was completed,excess HSiCl₃ was removed under vacuum.

3. A-[9]-Alkene (5)

A-[3]-SiCl₃ 4 was refluxed with 10% excess of allylmagnesium bromide indiethyl ether for 4 h, and cooled to 0° C. and hydrolyzed with 10%aqueous NH₄Cl. The organic layer was washed with water, dried MgSO₄ andconcentrated.

4. A-[9]-SiCl₃ (6)

A mixture of A-[9]-Alkene 5, HSiCl₃, and a common platinum-basedhydrosilylation catalyst, e.g. H2PtCl6 in propan-2-ol (Speier'scatalyst) or platinum divinylsiloxane complecx (Karstedt's catalyst),was stirred for 24 h at room temp. When the reaction was completed,excess HSiCl₃ was removed under vacuum.

Example 3.6 1. [1]-acid-[3]-triol (3)

(a) The triol 1 was cyanoethylated affording the nitrile compound 2.Acrylonitrile, nBu₃SnH, and azobisisobutyronitrile was added in PhCH₃including compound 1 at 110° C. (b) The nitirle compound 2 washydrolyzed to give compound 3 with carboxylic acid cleanly in suchcondition as KOH, EtOH/H₂O, H₂O₂, Δ.

2. A-[3]-triol (5)

(c) [1]-acid-[3]-triol was linked with compound 4 through an amidecoupling reaction using 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride (EDC) and 1-hydroxybenzotriazole hydrate (HOBT).

3. A-[3]-tribromide (6)

(d) The alcohol was used to synthesize tribromide by bromination withHBr/H₂SO₄ at 100° C.

4. [1]-CN-[3]-OBzl (8)

(e) The triol 1 was treated with benzyl chloride to give trisether usingMe₂SO and KOH. (f) The trisether 8 was cyanoethylated affording thenitrile compound 9. Acrylonitrile, nBu₃SnH, and azobisisobutyronitrilewas added in PhCH₃ including compound 8 at 110° C.

5. [1]-OH-[3]-OBzl (11)

(g) The nitirle compound 9 was hydrolyzed to give compound 10 withcarboxylic acid cleanly in such condition as KOH, EtOH/H₂O, H₂O₂, Δ. (h)The compound 10 with a carboxylic acid was proceeded with excess 1.0 MBH₃.THF solution to converse the acid into alcohol.

6. [1]-Alkyne-[3]-OBzl (13)

(i) The alcohol was transformed into chloride (CH₂Cl₂) with excess SOCl₂and a catalytic amount of pyridine. (j) The chloride was reacted withlithium acetylide ethylenediamine complex in dimethylsulphoxide at 40°C.

7. A-[3]-Alkyne-[9]-OBzl (14)

(k) The A-[3]-OBzl 6 was alkylated with 4 equivalents of terminal alkynebuilding block 13, hexamethylphosphoric rtriamide (HMPA), lithiumdiisopropylamide (LDA), and tetramethylethylenediamine (TMED) at 0-40°C. for 1.5 h.

Example 3.7 1. A-[9]-OH (15)

A-[3]-Alkyne-[9]-OBzl 14 was reduced and deprotected with Pd—C/H toproduce A-[9]-OH 15 in EtOH and THF solution including 10% Pd—C/H at 60°C. for 4 d.

2. A-[27]-COOH (17)

The alcohol was smoothly converted into the nonabromide employing SOBr₂in CH₂Cl₂ at 40° C. for 12 h. And then the nonabromide compound wasalkylated with 12 equivalents of [1]-Alkyne-[3]-OBzl 13 to give 49% ofA-[9]-Alkyne-[27]-OBzl 16. A-[9]-Alkyne-[27]-OBzl 16 were reduced anddeprotected in one step with Pd—C/H in EtOH and THF solution including10% Pd—C/H at 60□ for 4 d yielding 89% of A-[27]-OH. A-[27]-OH wasoxidized by RuO₄ treating with NH₄OH or (CH₃)₄NOH to achieve 85% ofA-[27]-COOH 17.

Example 3.8 1) [G1]-(OMe)₂ (3)

A mixture of compound 1 (1.05 mol equiv.), 3,5-dimethoxybenzyl bromide(1.00 mol equiv. 2), potassium carbonate (1.1 mol equiv.) and 18-c-6(0.2 mol equiv.) in dry acetone was heated at reflux under nitrogen for48 h. The mixture was cooled and evaporated to dryness, and the residuewas partitioned between CH₂Cl₂ and water. The aqueous layer wasextracted with CH₂Cl₂ (3×), and the combined organic layers were driedand evaporated to dryness. The crude product was purified by flashchromatography with EtOAc-CH₂Cl₂ as eluent to give compound 3.

2) [G1]-(OH)₂ (4)

Methyl ether group of compound 3 was deprotected by BBr₃ in EtOAcsolution for 1 h, and the crude product was purified by flashchromatography with MeOH-EtOAc as eluent to give compound 4.

3) [G2]-(OMe)₄ (5)

A mixture of [G1]-(OH)₂ (1.00 mol equiv. 4), 3,5-dimethoxybenzyl bromide(2.00 mol equiv. 2), potassium carbonate (2.1 mol equiv.) and 18-c-6(0.2 mol equiv.) in dry acetone was heated at reflux under nitrogen for48 h. The mixture was cooled and evaporated to dryness, and the residuewas partitioned between CH₂Cl₂ and water. The aqueous layer wasextracted with CH₂Cl₂ (3×), and the combined organic layers were driedand evaporated to dryness. The crude product was purified by flashchromatography with EtOAc-CH₂Cl₂ as eluent to give compound 5.

4) [G2]-(OH)₄ (6)

Methyl ether group of compound 5 was deprotected by BBr₃ in EtOAcsolution for 1 h, and the crude product was purified by flashchromatography with MeOH-EtOAc as eluent to give compound 4.

5) [G3]-(OMe)₈ (7)

A mixture of [G2]-(OH)₄ (1.00 mol equiv. 6), 3,5-dimethoxybenzyl bromide(4.00 mol equiv. 2), potassium carbonate (4.1 mol equiv.) and 18-c-6(0.2 mol equiv.) in dry acetone was heated at reflux under nitrogen for48 h. The mixture was cooled and evaporated to dryness, and the residuewas partitioned between CH₂Cl₂ and water. The aqueous layer wasextracted with CH₂Cl₂ (3×), and the combined organic layers were driedand evaporated to dryness. The crude product was purified by flashchromatography with EtOAc-CH₂Cl₂ as eluent to give compound 7.

6) [G3]-(OH)₈ (8)

Methyl ether group of compound 7 was deprotected by BBr₃ in EtOAcsolution for 1 h, and the crude product was purified by flashchromatography with MeOH-EtOAc as eluent to give compound 8.

Example 4 Assembly of the Dendron on a Substrate

TMAC (N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride) wasself-assembled on oxide glass instead of APDES. The dendrimer layer onTMAC layer did not need to cap the residual amine.

Aminosilylation with TMAC.

Clean substrates (slide glass) were placed into a solution of TMAC (2mL) and acetone (100 mL) for 5 h. After the self-assembly, thesubstrates were taken out of the flask, washed with acetone. Thesubstrates were placed in an oven, and heated at 110° C. for 40 min.After immersion in acetone, the substrates were sonicated for 3 min. Thewashed substrate was placed in a Teflon vessel, and placed in a glasscontainer with a big screw cap lined with an O-ring, and eventually thecontainer was evacuated (30-40 mTorr) to dry the substrate.

Structure of TMAC (N-trimethoxysilylpropyl-N,N,N-trimethylammoniumchloride)

Self-assembly of the Fmoc-spacer-[9]acid was performed in same conditionto the case of CBz-[9]acid with exception of capping of the residualamines by acetic anhydride

Self-Assembly of the Fmoc-Spacer-[9]acid (5).

A certain amount of the Fmoc-spacer-[9]acid (5) was dissolved in a mixedsolvent (DMF:deionized water=1:1 (v/v)) to make a solution of 20 mL. Thesolution was added into a Teflon vessel, and subsequently pieces of theabove prepared aminosilylated slide glass were placed in the solution.While allowing the flask at room temperature to self-assemble, eachpiece of the substrate was taken out of the solution after 1 day. Rightafter being taken out, the plate was washed with a copious amount ofdeionized water. Each substrate was sonicated for 3 min in deionizedwater, a mixture of deionized water-methanol (1:1 (v/v)), and methanolin a sequential manner. After sonication, the substrates were placed ina Teflon vessel, and placed in a glass container with a big screw caplined with an O-ring, and eventually the container was evacuated (30-40mTorr) to dry the substrate.

Deprotection of Fmoc from the Self-Assembled Fmoc-Spacer-[9]acid (5).

Teflon vessels containing 5% piperidine in DMF were prepared. Theself-assembled substrates were immersed in the vessels, and stirred for20 min. Each substrate was sonicated for 3 min in acetone, and MeOH in asequential manner and evacuated in a vacuum chamber (30-40 mTorr).

Example 5 Preparation of Dendron-Modified AFM Tip and Substrate

Materials

The silane coupling agentN-(3-(triethoxysilyl)propyl)-O-polyethyleneoxide urethane (TPU) waspurchased from Gelest Inc. All other chemicals are of reagent grade fromSigma-Aldrich. The UV-grade fused silica plates were purchased from CVILaser Co. The polished prime Si(100) wafers (dopant, phosphorus;resistivity, 1.5-2.1 Ω·cm) were purchased from MEMC Electronic MaterialsInc. Deionized water (18 MΩ·cm) was obtained by passing distilled waterthrough a Barnstead E-pure 3-Module system. Thickness was measured witha variable angle ellipsometer (Model M-44) from J. A. Woolam Co. UV-visspectra were recorded with a Hewlett-Packard diode array 8453spectrophotometer.

1) Cleaning the Substrates.

Fused silica plates and silicon wafers were sonicated in Piranhasolution (concentrated H₂SO₄:30% H₂O₂=7:3 (v/v)) for 4 h (Caution:Piranha solution can oxidize organic materials explosively. Avoidcontact with oxidizable materials.). The plates and the wafers werewashed and rinsed thoroughly with deionized water after the sonication.Subsequently, the substrates were immersed in a mixture of deionizedwater, concentrated ammonia solution, and 30% hydrogen peroxide (5:1:1(v/v/v)) contained in a Teflon beaker. The beaker was placed in a waterbath and heated at 80° C. for 10 min. The substrates were taken out ofthe solution and rinsed thoroughly with deionized water. Again, thesubstrates were placed in a Teflon beaker containing a mixture ofdeionized water, concentrated hydrochloric acid, and 30 % hydrogenperoxide (6:1:1 (v/v/v)). The beaker was heated at 80° C. for 10 min.The substrates were taken out of the solution and washed and rinsedthoroughly with a copious amount of deionized water. The cleansubstrates were dried in a vacuum chamber (30-40 mTorr) for about 20 minand used immediately in the following steps.

2) Cleaning the Tip.

The standard V-shaped silicon nitride cantilevers (MLCT-AUNM) withpyramidal tips (Veeco Instrument; k=10 pN/nm) were first activated bydipping in 10% nitric acid and heating at 80° C. for 20 min. Thecantilevers were taken out of the solution and washed and rinsedthoroughly with a copious amount of deionized water. The cleancantilevers were dried in a vacuum chamber (30-40 mTorr) for about 20min and used immediately in the following steps.

3) Aminosilylation.

Clean fused silica, silicon wafer, and cantilevers were immersed intoanhydrous toluene (20 mL) containing the coupling agent (0.20 mL) undernitrogen atmosphere, and placed in the solution for 6 h. Aftersilylation, the substrates and cantilevers were washed with toluene,baked for 30 min at 110° C. The substrates were immersed in toluene,toluene-methanol (1:1 (v/v)), and methanol in a sequential manner, andthey were sonicated for 3 min in each washing solution. The cantileversrinsed thoroughly with toluene and methanol in a sequential manner.Finally the substrates and cantilevers were dried under vacuum (30-40mTorr).

4) Preparation of Dendron Modified Surface.

The above hydroxylated substrates and cantilevers were immersed into amethylene chloride solution with a small amount of DMF dissolving thedendron (1.0 mM) and a coupling agent, 1,3-dicyclohexylcarbodiimide(DCC) (9.9 mM) in the presence of 4-dimethylaminopyridine (DMAP) (0.90mM) for 12˜24 h. The dendron (9-anthrylmethylN-({[tris({2-[({tris[(2-carboxyethoxy)methyl]methyl}amino)carbonyl]ethoxy}methyl)methyl]amino}carbonyl)propylcarbamate)used in this work was prepared in this group. After reaction, thesubstrates were immersed in methylene chloride, methanol, and water in asequential manner, and they were sonicated for 3 min at each washingstep. The cantilevers were rinsed thoroughly with methylene chloride,methanol, and water in a sequential manner. Finally the substrates andcantilevers were washed with methanol, and dried under vacuum (30-40mTorr).

Example 6 Immobilization of Oligonucleotides

1) Deprotection of Carboanthrylmethoxy Group from the Dendron Surface.

The dendron modified substrates and cantilevers were immersed into amethylene chloride solution with 1.0 M trifluoroacetic acid (TFA), andthey were stirred for 3 h. After the reaction, they were soaked in amethylene chloride solution with 20% (v/v) diisopropylethylamine (DIPEA)for 10 min. The substrates were sonicated in methylene chloride andmethanol each for 3 min and the cantilevers were rinsed thoroughly withmethylene chloride and methanol in a sequential manner. The substratesand cantilevers were dried under vacuum (30-40 mTorr).

2) Preparing the NHS-Modified Substrates.

The above deprotected substrates and cantilevers were immersed into anacetonitrile solution with di(N-succinimidyl)carbonate (DSC) (25 mM) andDIPEA (1.0 mM) for 4 h under nitrogen atmosphere. After the reaction,the substrates and cantilevers were placed in stirred dimethylformamidefor 30 min and washed with methanol. The substrates and cantilevers weredried under vacuum (30-40 mTorr).

3) Immobilization of Oligonucleotides on the Dendron ModifiedSubstrates.

The above NHS-modified substrates and cantilevers were soaked in anoligonucleotide (20 μM) in 25 mM NaHCO₃ buffer (pH 8.5) with 5.0 mMMgCl₂ for 12 h. After the reaction, the substrates and cantilevers werestirred in a hybridization buffer solution (2×SSPE buffer (pH 7.4)containing 7.0 mM sodium dodecylsulfate) at 37° C. for 1 h and inboiling water for 5 min to remove non-specifically boundoligonucleotide. Finally the substrates and cantilevers were dried undervacuum (30-40 mTorr). The oligonucleotides to be immobilized are shownin Table 1.

Example 7 AFM Force Measurements 7-1: Sample Preparation

To understand effect of the spacing, the two types of the modification(9-acid/GPDES substrate and 9-acid/TPU substrate) were employed for thesubstrate by using the two silane agents such as GPDES and TPU, whilespacing on AFM tip was fixed with use of 9-acid/TPU. The surfacemodification of the substrate was performed according to Examples 1. Theoligonucleotides as shown in SEQ ID NOs: 1 to 4 were immobilized on the9-acid/TPU substrate, respectively according to Example 2. The 30 bpcomplementary DNA as represented by SEQ ID NO: 2 was immobilized on the9-acid/GPDES substrate. The oligonucleotides as shown in SEQ ID NOs: 5to 20 were immobilized on the 9-acid/TPU type of AFM tip, respectively.

TABLE 2 Immobilized Type of nucleotide Surface oligonucleotide (SEQ IDNO) AFM tip 9-acid/TPU Perfect match DNA 5 to 8 9-acid/TPU 1 bp mismatch 9 to 12 9-acid/TPU 2 bp mismatch 13 to 16 Substrate 9-acid/GPDES DNA 1to 4 9-acid/TPU DNA 1 to 4 27-acid/TPU DNA 1 to 4

In the example, 9-acid dedron is (9-anthrylmethylN-({[tris({2-[({tris[(2-carboxyethoxy)methyl]methyl}amino)carbonyl]ethoxy}methyl)methyl]amino}carbonyl)propylcarbamate),and 27-acid dedron is described in Example 3.

7-2: AFM Force Measurement

All force measurements were performed with a NanoWizard AFM (JPKInstrument). The spring constant, k_(c), of each individual AFM tip wascalibrated in solution before each experiment by thermal fluctuationmethod available via a NanoWizard software. The spring constant variedbetween 12 and 15 pN/nm. All measurements were carried out in a freshPBS buffer (pH 7.4) at room temperature. The loading rate of forcemeasurements varied between 110 nm/s and 540 nm/s. At each experimentalcondition, force curves were recorded more than one hundred times at aspot, and at least more than 5 spots were examined. In thesemeasurements, both binding and unbinding force curves were recorded. Tocalculate distance that the tip actually moved, the cantileverdisplacement was subtracted from the piezo displacement. The cantileverdisplacement was obtained by dividing the force by the cantilever springconstant.

7-3: Unbinding Force for 9-Acid/GPDES Substrate Immobilized by aComplementary 30-Base Pair DNA

Using the oligonucleode as shown in SEQ ID NO: 2 immobilized on9-acid/GPDES substrate, and the oligonucleode as shown in SEQ ID NO: 6immobilized on 9-acid/TPU AFM tip, AFM force measurement was performedat various loading rate in the range between 110 nm/s and 540 nm/saccording to AFM measurement of example 3-2 to obtain unbinding forcedistribution (FIG. 4A) at a retraction rate of 110 nm/s, and forcedistance curve (FIG. 4B) and unbinding force distribution (FIG. 4C) at aretraction rate of 540 nm/s.

A large unbinding force, attributable to an interaction of multipleoligonucleotides, was observed at 540 nm/s retraction rate (FIG. 4B).Also, the histogram is rather broad (the maximum half-width is 15 pN.)and unresolved (FIG. 4C). However, at 110 nm/s refraction rate thehistogram (FIG. 4A) was resolved into three peaks, and each peak wassharp (the maximum half-width is 3 pN for the first peak.). Exactinterpretation of the behavior is not straightforward, but the firstpeak at 37 pN is very likely to be from single DNA-DNA interaction (videinfra) and the other two (46 pN and 55 pN) represent unbinding eventswith the secondary interaction in addition to the single one.

FIG. 4A is a histogram showing the force distribution of a complementary30-base pair when relatively narrow spacing (realized with a dendron onthe GPDES substrate). FIG. 4B is a direct measurement of singleunbinding force of complementary 30 base pairs with a retractionvelocity of 540 nm/s. FIG. 4B is a force versus distance curve measuredbetween complementary 30 base pairs with a retraction velocity of 540nm/s. Much larger force (blue curve), attributable to interactions ofmultiple oligonucleotides, can be observed at 540 nm/s retraction rate(For comparison, unbinding force (red curve) observed in 110 nm/sretraction rate is displayed.). FIG. 4C shows the probabilitydistribution of unbinding force with a retraction velocity of 540 nm/s.The histogram shows the observed force distribution with relativelynarrow spacing (realized with the dendron on the GPDES surface). Themaximum of the distribution is found by a Gaussian fit to be 68±13 pN,and the distribution curve is not resolved to show single interaction.

7-4: Binding Force and Unbinding for 9-Acid/TPU Substrate Immobilized bya Complementary 30-Base Pair DNA

Using the oligonucleode as shown in SEQ ID NO: 2 immobilized on9-acid/TPU substrate, and the oligonucleode as shown in SEQ ID NO: 6immobilized on 9-acid/TPU AFM tip, AFM force measurement was performedat a retraction rate of 110 nm/s according to AFM measurement of example3-2 to obtain unbinding force distribution (FIG. 5A), binding force vsdistance curve (FIG. 5B), and binding force distribution curve (FIG.5C).

When the DNA was immobilized on 9-acid/TPU surface, the unbinding forcehistogram (FIG. 5A) showed only one peak at 37±2 pN, and the narrownessof the peak was not tarnished. Disappearance of the minor peaks at 46 pNand 55 pN confirms that these peaks represent events associated with thesecondary interaction. For analysis of the above two cases, only unusualcurves were discarded, and more than 90% of measurements were includedin the plot. While the curves are frequently indented for 9-acid/GPDEScase, none of the curves for 9-acid/TPU showed any indentation. Thus, itis possible to measure single DNA-DNA interaction by modifying thesubstrate surface with TPU as a silane agent, because of the sufficientspacing.

The binding force curves were observed every time when the tipapproached the dendron-modified surface (FIG. 5B).

In this particular process, again 9-acid/TPU-modified surface producedsingle dip force curves, while 9-acid/GPDES case frequently showeddouble- or multiple-dipped force curves. Because the behavior was soconsistent and reproducible, not a single datum had to be discarded togenerate the histogram. As in the histogram for the unbinding event, thepeak is narrow (the maximum half-width is 3 pN.), and the value of 39 pNis pretty close to that of the unbinding case. It is intriguing to findthat such unprecedented binding process can be observed when the spacingbetween DNAs was controlled properly.

Moreover, it was found that the binding force behavior is less dependenton the loading rate. In other word, the same histogram (FIG. 5C) wasobtained at any loading rate between 70 nm/s and 540 nm/s. Theparticular experiment was repeated many times using different tips andsamples, and the above binding behavior and the histogram wereconsistently reproduced.

7-5: Unbinding Force for 27-Acid and TPU Modified Substrate forExamining Single Strand Interaction

Previously, unbinding force of 48 pN for other complementary 30-base DNAwas recorded even at a slower retraction rate (T. Strunz, K. Oroszlan,R. Schäfer, H.-J. Güntherodt, Proc. Natl. Acad. Sci. U.S.A. 96, 11277,1999). It is interesting to observe a smaller unbinding force even withDNA with the same GC content.

In order to examine whether these interactions are from a single strandor multiple strands, a higher generation dendron, 27-acid, was employed.The third generation dendron is expected to provide spacing around 10nm. Spacing on the substrate was increased with a combination of 27-acidand TPU, while AFM tip was modified with 9-acid/TPU. It was interestingto note the histogram was exactly same as that of 9-acid/TPU case. TheAFM tip was modified with 27-acid/TPU, and observed again the samehistogram. The only difference is a reduced chance of observing theunbinding. For the last case, about 50% of the refraction events did notshow the unbinding phenomenon at all. This change seems reasonable,because too big spacing between the oligonucleotides reduces chance ofthe hybridization. This behavior showed clearly spacing generated from9-acid/TPU was already large enough for realization of single strandinteraction.

7-6: Binding Force and Unbinding Force for Complementary DNA Duplexes

Prior to testing other oligonucleotides, the accuracy of the forcemeasurement was tested in the above condition. Samples from differentbatches were prepared, and the cantilevers were calibrated. Theinventors found that the variation was within 10-15%. The value suggeststhat reproducible and precise control of the surface allows minimalerror: the value never goes beyond the error associated with springconstant calibration. With 9-acid/TPU-modified surface, binding andunbinding events of DNA duplexes of 20, 30, 40, and 50 base pairs(Table 1) were performed at the 110 nm/s loading rate. Force-distancecurves were obtained on each duplex during the approach and retractcycles.

As previously mentioned above, binding and unbinding histograms werealmost the same, and average force values were identical. The bindingforce histogram, and the unbinding force histogram of complementary DNAduplexes with 20, 30, 40 and 50 base pairs, were shown in FIG. 6A, andFIG. 6C, respectively.

In the histogram as shown in FIG. 6A, non-overlapped peaks, and clearincrement of the force with the length of DNA were seen. The values are29 pN, 39 pN, 50 pN, and 59 pN for 20, 30, 40, and 50 base pairs.Coincidentally the increment of the force is roughly 10 pN at eachincrease of 10 DNA bases. For verification, the forces ofnon-complementary DNA strands were measured. In all cases, force curveswere mostly not detected, and with a low probability, a tiny force of 10pN was recorded.

In FIG. 6B for force-piezo displacement curve of complementary DNAduplexes with 20, 30, 40 and 50 base pairs was obtained by calculatingfrom the binding force distribution of FIG. 4. The observed distancethat the tip moved towards the surface to relieve the strain upon thebinding event was retrieved from the force-piezo displacement curve andthe value was plotted. In the particular situation, distances of 2.4 nm,3.2 nm, 3.6 nm, and 4.2 nm were recorded for 20-mer, 30-mer, 40-mer, and50-mer cases. Because the peaks are quite narrow, and the distanceincreases almost linearly with the DNA length, the parameter should bediagnostic for analyzing the interaction DNA length in unknown samples.

7-7: Binding Force Distribution for Mismatched DNA Duplexes

To further probe this recognition phenomenon, interaction force curveswere recorded for the single base and double base mismatched pairs(Table 1). Using the oligonucleodes as shown in SEQ ID NO: 5 to 8immobilized on 9-acid/TPU substrate for single base mismatched DNA, theoligonucleodes as shown in SEQ ID NO: 9 to 12 immobilized on 9-acid/TPUsubstrate for double base mismatched DNA, and the oligonucleode as shownin SEQ ID NO:1 to 4 immobilized on 9-acid/TPU AFM tip, AFM forcemeasurements were performed at a retraction rate of 110 nm/s accordingto AFM measurement of example 3-2 to obtain binding force distributionfor single base mismatched DNA duplexs (FIG. 7), and binding forcedistribution for double base mismatched DNA duplexs (FIG. 8).

As expected, it was observed that the introduction of the mismatchdecreased binding and unbinding forces. As shown in FIG. 7 for singlebase mismatched pairs, binding force of 27 pN, 37 pN, 43 pN, and 50 pNwas observed for 20-mer, 30-mer, 40-mer, and 50-mer, respectively. Asshown in FIG. 8 for double base mismatched pairs, binding force of 24pN, 32 pN, 40 pN, and 45 pN was observed for 20-mer, 30-mer, 40-mer, and50-mer, respectively.

As in the previous case for complementary DNA duplex, binding andunbinding forces were identical. However, for single base mismatchcases, there were only marginal decrease (2 pN) for both 20 mer and 30mer. Meanwhile, substantial decrease (>7 pN) was observed for 40 mer and50 mer. The result shows that use of DNA longer than 40 mer guaranteesreliable detection of single point mutation. As expected, largerreduction of the force was observed for the double base mismatchedpairs. For examples, decrease of 5 pN was observed for 20 mer, while 14pN was observed for 50 mer. It is worthwhile to note the capability ofpicoforce AFM for detecting a single point mutation at the singlemolecular level.

Example 8 Biomolecular Interaction Between Signal Transducer Proteins

Previous studies showed that a dendron-modified surface can ensureenough spacing between biomolecules attached on surface by controllingthe size of the dendron molecule. In this study, an AFM tip and a solidsubstrate such as a Si wafer were also functionalized with a dendronmolecule, as described in the previous published paper (Langmuir 2005,21, 4257) and U.S. patent application Ser. No. 10/917,601, to improvethe recognition efficiency between proteins at the single molecularlevel (FIG. 11). The dendron molecule used here is the same as theprevious one (Langmuir 2005, 21, 4257), but all dendron moleculesreferenced in U.S. patent application Ser. No. 10/917,601 can beutilized for this study.

After the deprotection of a protecting group, the dendron-modifiedsubstrate was incubated in a 50 mM NaHCO₃ buffer (pH 8.5) with a smallamount of DMF dissolving N-succinimidyl-4-maleimidobutylate (GMBS) (16mM). After 3 h incubation at room temperature, it was rinsed thoroughlywith D.I. water. The GMBS-coated substrate was immersed in PBS buffersolution (10 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl, pH 7.4) withglutathione (GSH) (16 mM) for 12 h, and then was sonicated in D.I. waterfor 3 min. After being immersed in PBS buffer solution with2-mercaptoethanol (1.6 M) for 2 h to quench the remained active GMBSfunctional groups, it was sonicated in D.I. water for 3 min. Next, theGSH-coated one was incubated in PBS buffer containing 0.43 μg/mlGST-tagged PLD1-PX at 4° C. for 30 min, and then was rinsed with PBSTbuffer (10 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl, 0.1% tween 20,pH 7.4). Finally, it was stored in PBS buffer at 4° C. for furtherstudy.

A silicon nitride (Si₃N₄) AFM tip was immersed in HNO₃:H₂O (3:1 (v/v))solution, and after being heated at 80° C. for 20 min, the tip waswashed with D.I. water. The modification of the cleaned tip was the sameas that of the Si wafer substrate except the introduction of GST-taggedMunc-18-1. The GSH-coated tip was incubated in PBS buffer solution with0.97 μg/ml GST-tagged Munc-18-1 at 4° C. for 30 min. The final coatedtip was rinsed with PBST buffer solution and stored at 4° C. for furtherstudy.

Both Munc-18-1 and PLD1-PX were signal transducer proteins and it hasbeen known that Munc-18-1 in a cytoplasm of a brain cell forms a complexwith phospholipase D1 (PLD1-PX) through PX domain of PLD1-PX in vivo.

When an AFM tip coated with Munc-18-1 approaches closely onto asubstrate coated with PLD1-PX, and then retreats back, the binding forcebetween both proteins can be measured (FIG. 12). The measured force isconstant around 50 pN which means that one Munc-18-1 molecule interactedwith one PLD1-PX in this study (FIG. 14( a)). Furthermore, whenA-[27]-acid instead of A-[9]-acid was used as a dendron, it was observedthat the ratio of single interaction to multiple one was enhanced from1.5:1 to 3:1. This result implies that the bigger size of biomoleculesneed the larger spacing between them on surface for the specific singleinteraction, and this need can be easily satisfied by utilizingsize-controllable dendron molecules. For additional study, in order toprove that the measured force results from the specific interactionbetween Munc-18-1 and PLD1-PX, not non-specific one, excess amounts offree Munc-18-1 in solution were added and blocked the binding site ofPLD1-PX during the force measurement (FIG. 13( a)). As a result, theinteraction force between Munc-18-1 on the tip and PLD1-PX on thesubstrate was not observed (FIG. 14( b)). Therefore, these above resultsshows that Munc-18-1 binds to PLD1-PX specifically with a constant forceand a dendron-modified surface can control the spacing amongbiomolecules on surface resulting in the specific single biomolecularinteraction.

Example 9 Biomolecular Interaction Among Three Different Biomoleculesand its Application to a Drug Screening

Some human diseases result from undesirable interaction between proteinsin a body and several drug screening methods have been utilized to findthe best drug candidates for those diseases. Recently, a Bio-AFM hasalso been applied to the drug screening assay. This method determinesdrug efficiency by measuring the interaction force between two differentproteins before and after adding a drug candidate. In addition, it ispossible to detect easily the optimal drug concentration for a medicaltreatment by controlling the drug concentration in situ. Here, we showthat the dendron-modified surface can be applicable to a drug screeningwith a Bio-AFM.

In this study, Munc-18-1 and PLD1-PX were introduced each onto an AFMtip and a substrate such as a Si wafer, as described in EXAMPLE 8. Adrug candidate is PLC-γ1 which binds to PLD1-PX on the solid substrateand competes against Munc-18-1 on the tip (FIG. 13( b)). As testing withseveral different concentrations of PLC-γ1, high concentration of PLC-γ1prevented PLD1-PX from binding to Munc-18-1 perfectly, but lowconcentration did not (FIG. 15). Consequently, this study suggests thatPLC-γ1 is a competitor of Munc-18-1 and the interaction force betweenPLD1-PX and Munc-18-1 depends on the concentration of PLC-γ1. Therefore,this Bio-AFM assay can be extended to research for dug discovery andmedical treatment.

Example 10 Biomolecular Interaction Between a Streptavidin and a Biotin

Streptavidin and a biotin have been used widely as a simple biomolecularinteraction model. Here, this simple model was applied to a researchfield of force measurement with Bio-AFM. Because a streptavidin has twobinding sites on its one side, it is difficult to prove that themeasured force by a Bio-AFM result from the interaction between onestreptavidin and one biotin. However, by controlling the spacing amongbiomolecules with a dendron molecule, we can observe one to oneinteraction force more easily.

An AFM tip and a solid substrate such as a Si wafer were functionalizedwith a dendron molecule, as described in EXAMPLE 8. A streptavidin wasattached onto the substrate and a biotin onto the tip through DSC(di(N-succinimidyl)carbonate) linker molecule. The measured force isconstant and almost similar to the published value which is believed toresult from the interaction between one streptavidin and one biotin.This result suggests that a biotin molecule binds to a streptavidinmolecule specifically through single interaction on the dendron-modifiedsurface.

Moreover, it was observed that the ratio of single interaction tomultiple one was higher on the surface coated with A-[9]-acid thanA-[3]-acid. Namely, because of the small size of A-[3]-acid, the spacingbetween biomolecules on surface was not enough for one to oneinteraction. Consequently, because a dendron-modified surface cancontrol the spacing among biomolecules on surface, it can ensure thespecific single biomolecular interaction.

Example 11 Mapping of a Specific Ligand on a Cell Surface ThroughBiomolecular Interaction Between a Peptide and a Protein

Although the interaction between a receptor on a cell surface and aligand has been studied in depth using a confocal microscopy, thismethod has a unsolved problem that each receptor on a cell couldn't bedefined with high resolution in nanometer scale. Recently, a Bio-AFMinstrument has been made to overcome this limit. However, in spite ofshowing the possibility to define each receptor individually, thismethod also has an unsatisfied issue which is non-specific binding of anAFM tip to a cell surface and multiple binding between ligands on a tipand receptors on a cell. These problems can provide wrong informationfor receptor distribution on a cell. Previous studies showed that adendron-modified surface has the characteristic of low non-specificbinding with biomolecules and ensure the single biomolecular interactionby providing enough spacing between biomolecules attached on thesurface. Therefore, the dendron-modified surface can easily help eachreceptor be detected separately with high resolution by a Bio-AFM.

RBL2H3 cell used in this study had an overexpressed FPR1 (Formyl PeptideReceptor 1) which is related to inflammation. After being cultivated inDMEM (10% FBS, 1% penicillin/streptomycin) under 5% CO₂ condition at 37°C. for 48 h, the cell with 5×10⁴ cells/ml was attached onto a coverglass using 5% Matrigel solution. The ligand binding to FPR1 is thesynthetic peptide consisting of six amino acids which cause inflammationand has a cysteine at its N terminal position for linking with GMBSlinker molecule like the below. In addition, the peptide is neutralizedin charge through acetylation at N terminal and amidation at C terminal.

Ac-Cysteine-linker-WKYMVm-NH₂

An AFM tip was modified with a dendron molecule, as described in EXAMPLE8. After being functionalized with GMBS and the peptide ligand, the tipscanned the cell surface measuring the force between a receptor and aligand on a certain area of the cell (FIG. 16( a)). The experiment wasperformed in 1×PBS buffer solution (pH 7.4) at room temperature usingThe Nano Wizard® Atomic Force Microscope (JPK Instruments, Inc) as aBio-AFM.

FIG. 16 shows some of the measured force graphs where a blue line meansthe backward force curve as retracting a tip. From this curve, eachforce can be calculated and finally combined to make a force map torepresent the distribution of the receptor on the cell surface. FIG. 17shows the force map and force histogram for the interaction between FPR1and its ligand peptide. A bright pixel means a strong force on the map,while a dark pixel does a weaker one (FIGS. 17 & 18). Two remarkableforces, 31 pN and 55 pN, were observed from the force histogram (FIG.17). For a further study, an additional experiment was performed with acompetitor, free WKYMVm in solution in order to prove that the measuredforce definitely came from the specific interaction between FPR1 and itsligand peptide (FIG. 18). As a result, it was found that the populationof the force around 60 pN decreased significantly after incubation withthe free peptide, WKYMVm (SEQ ID NO:17) for 1 h. This result suggeststhat the force around 60 pN results from the specific interactionbetween the receptor and the ligand, but the force around 30 pN is thebackground force due to the non-specific interaction.

Example 12 Biomolecular Interaction Between a Protein and a Glycolipid

Cholera toxin B has been known to bind selectively to one ofglycolipids, ganglioside GM1 which exists on the surface of a humansplanchnic epithelial cell and then to give rise to pain. Here we studythe distribution of a ganglioside GM1 on a cell surface by measuring theforce with its ligand, cholera toxin B.

A test cell is human epithelium with the overexpressed ganglioside GM1.After being cultivated in DMEM (10% FBS, 1% penicillin/streptomycin)under 5% CO₂ condition at 37° C. for 48 h, the cell with 5×10⁴ cells/mlis attached onto a cover glass using 5% Matrigel solution. An AFM tip ismodified with a dendron molecule followed by the deprotection of aprotecting group, as described in EXAMPLE 8. After being functionalizedwith a linker molecule and cholera toxin B, the tip scans the cellsurface measuring the force between a receptor and ligand on a certainarea of the cell. The experiment is performed in 1×PBS buffer solution(pH 7.4) at room temperature using The NanoWizard® Atomic ForceMicroscope (JPK Instruments, Inc) as a Bio-AFM.

Example 13 Biomolecular Interaction Between a Lectin and a Glycoprotein

Concanavalin A, one of lectin proteins, has been used widely forstudying the characteristics of a glycoprotein because it binds to aglycoprotein strongly. Here we investigate the distribution of aglycoprotein having a mannose at its terminal position, usingconcanavalin A as a ligand. The cell used in this experiment is afibroblast with the glycoprotein on its surface.

After being cultivated in RPMI (10% FBS, 1% penicillin/streptomycin)under 5% CO₂ condition at 37° C. for 48 h, the cell with 5×10⁴ cells/mlis attached onto a cover glass using 5% Matrigel solution. An AFM tip ismodified with a dendron molecule followed by the deprotection of aprotecting group, as described in EXAMPLE 8. After being functionalizedwith a linker molecule and Concanavalin A, the tip scans the surface ofthe cell attached on a cover glass measuring the force between areceptor and ligand on a certain area of the cell. The experiment isperformed in 1×PBS buffer solution (pH 7.4) at room temperature usingThe Nano Wizard® Atomic Force Microscope (JPK Instruments, Inc) as aBio-AFM.

Example 14 Biomolecular Interaction Between a Carbohydrate and aGlycoprotein

Mycobacterium tuberculosis causes pulmonary tuberculosis because itssurface has a HBHA (heparin-binding haemagglutinin adhesin) which bindsto a heparin on an epithelial cell of a lung. Here we study thedistribution of a HBHA on the surface of Mycobacterium tuberculosis,using a heparin as a ligand. The cell used in this experiment is amycobacterium bovis BCG cell with a HBHA on its surface.

After being cultivated in Sauton medium under 5% CO₂ condition at 37° C.for 48 h, the cell with 5×10⁴ cells/ml is attached onto a cover glassusing 5% Matrigel solution. An AFM tip is modified with a dendronmolecule followed by the deprotection of a protecting group, asdescribed in EXAMPLE 8. After being functionalized with a linkermolecule and a heparin, the tip scans the surface of the cell attachedon a cover glass measuring the force between a receptor and ligand on acertain area of the cell. The experiment is performed in 1×PBS buffersolution (pH 7.4) at room temperature using The NanoWizard® Atomic ForceMicroscope (JPK Instruments, Inc) as a Bio-AFM.

Example 15 Biomolecular Interaction Between a Nerve Growth Factor (NGF)and a Tyrosine Kinase A

NGF has been known to bind to a TrkA (tyrosine kinase A) on the surfaceof a nerve cell and control its survival function. In this study, weinvestigate the distribution of a TrkA expressed on the surface of apheochromocytoma PC12 cell, using a NGF as a ligand.

After being cultivated in RPMI (5% FCS, 1% penicillin/streptomycin)under 5% CO₂ condition at 37° C. for 48 h, the cell with 5×10⁴ cells/mlis attached onto a cover glass using 5% Matrigel solution. An AFM tip ismodified with a dendron molecule followed by the deprotection of aprotecting group, as described in EXAMPLE 8. After being functionalizedwith a linker molecule and the ligand, NGF, the tip scans the surface ofthe cell attached on a cover glass measuring the force between areceptor and ligand on a certain area of the cell. The experiment isperformed in 1×PBS buffer solution (pH 7.4) at room temperature usingThe Nano Wizard® Atomic Force Microscope (JPK Instruments, Inc) as aBio-AFM.

While this invention has been described in connection with what ispresently considered to be practical exemplary embodiments, it is to beunderstood that the invention is not limited to the disclosedembodiments, but, on the contrary, is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

1-30. (canceled)
 31. A method for determining the presence of amismatched nucleotide between a probe nucleotide and a targetnucleotide, said method comprising: comparing a control atomic forcemicroscopy (AFM) binding force with a measured AFM binding force betweena probe nucleotide and a target nucleotide to determine whether there isa mismatch between said probe nucleotide and said target nucleotide,wherein said probe nucleotide is attached to a cantilever of said AFMvia a dendron comprising: branched regions, and a single linear region,and wherein a plurality of termini of said branched regions of dendronare covalently bound to the surface of said cantilever, and said probenucleotide is attached to the terminus of the single linear region ofsaid dendron.
 32. The method of claim 31, wherein said control AFMfinding force comprises a AFM binding force between said probenucleotide and a perfectly matching oligomer, and wherein the presenceof a difference in the control AFM binding force and the measured AFMbinding force is an indication that a mismatched nucleotide is presentbetween said probe nucleotide and said target nucleotide.
 33. The methodof claim 31, wherein the target nucleotide comprises a portion of agene, and wherein the presence of mismatched nucleotide is an indicationthat a mutation is present in said gene.
 34. The method of claim 31,wherein the control AFM binding force comprises AFM binding forcesbetween said probe nucleotide and a plurality of mismatchedoligonucleotides.
 35. The method of claim 34, wherein said step ofcomparing the control AFM binding force with the measured AFM bindingforce comprises comparing the measured AFM binding force with AFMbinding forces between said probe nucleotide and a plurality ofmismatched oligonucleotides.
 36. The method of claim 35, wherein saidstep of comparing the measured AFM binding force with the control AFMbinding force is used to determine the number of mismatched nucleotidesbetween said probe nucleotide and said target nucleotide.
 36. The methodof claim 35, wherein said step of comparing the measured AFM bindingforce with the control AFM binding force is used to determine thelocation of mismatched nucleotide between said probe nucleotide and saidtarget nucleotide.
 37. The method of claim 31, wherein the presence of amismatched nucleotide between said probe nucleotide and said targetnucleotide is an indication of the presence of a disease.
 38. The methodof claim 31, wherein said method measures AFM binding force between asingle target nucleotide and said probe nucleotide.
 39. The method ofclaim 31, wherein said target nucleotide is bound to a substratesurface.
 40. The method of claim 39, wherein said target nucleotide isbound to the substrate surface via a dendron comprising: branchedregions, and a single linear region, and wherein a plurality of terminiof said branched regions of dendron are covalently bound to the surfaceof said cantilever, and said probe nucleotide is attached to theterminus of the single linear region of said dendron.